阅读说明：本技术 Fam19a5的医药用途 (Medical application of FAM19A5 ) 是由 王应 马大龙 黄诗扬 邢国刚 宋占明 梁炜薇 佘少平 李青青 刘中天 于 2021-03-29 设计创作，主要内容包括：本发明公开了FAM19A5蛋白在抑郁症、认知障碍、神经炎症的诊断、预防或治疗中的医药用途。FAM19A5蛋白具有调节抑郁及认知相关行为的作用,FAM19A5蛋白、编码FAM19A5的多核苷酸、含有该多核苷酸的基因工程载体和/或宿主细胞,以及药物组合物,可应用于预防、诊断或治疗抑郁症、认知障碍、神经炎症,还可以以所述蛋白或多核苷酸作为靶标开发化合物、抗体、多肽药物和商品化试剂,对相关疾病进行研究。(The invention discloses medical application of FAM19A5 protein in diagnosis, prevention or treatment of depression, cognitive disorder and neuroinflammation. The FAM19A5 protein has the functions of regulating depression and cognition related behaviors, the FAM19A5 protein, polynucleotide for coding FAM19A5, genetic engineering vector and/or host cell containing the polynucleotide and a pharmaceutical composition can be applied to prevention, diagnosis or treatment of depression, cognitive disorder and neuroinflammation, and the protein or the polynucleotide can be used as a target to develop compounds, antibodies, polypeptide drugs and commercial reagents to research related diseases.)

Use of FAM19a5 or a homologous protein thereof for the manufacture of a medicament for the prevention and/or treatment of a disease, wherein the disease comprises depression, cognitive disorders, neuroinflammation; the FAM19A5 or homologous protein thereof is:

(1) has the sequence shown in SEQ ID No: 2 or SEQ ID No: 3; or

(2) A protein having at least 80% amino acid sequence homology with the protein of (1), and having the same or similar biological functions as the protein of (1).

2. Use of a polynucleotide encoding FAM19a5 or a homologous protein thereof for the manufacture of a medicament for the prevention and/or treatment of a disease, wherein the disease comprises depression, cognitive disorders, neuroinflammation; the polynucleotide is:

(i) encoding SEQ ID No: 2 or SEQ ID No: 3; or

(ii) (ii) a polynucleotide having at least 80% sequence homology to the polynucleotide of (i) which encodes a protein having the same or similar biological function as the protein encoded by the polynucleotide of (i).

3. The use of claim 2, wherein the polynucleotide has the sequence set forth in SEQ ID No:1 is shown.

4. Use of a genetically engineered vector comprising a polynucleotide according to claim 2 or 3 in the manufacture of a medicament for the prevention and/or treatment of a disease, wherein the disease comprises depression, cognitive disorders and neuroinflammation.

5. The use of claim 4, wherein the genetically engineered vector is an adeno-associated virus comprising a neuron-specific promoter.

6. Use of FAM19a5 or a homologous protein thereof or polynucleotides encoding same as claimed in claim 1 for the preparation of commercial reagents for the study of diseases, including depression, cognitive disorders and neuroinflammation.

7. The use of claim 6, wherein said commercial agent comprises FAM19A5 or a homologous protein thereof or a polynucleotide encoding same, or a compound, antibody, polypeptide or oligonucleotide that targets said FAM19A5 or a homologous protein thereof or a polynucleotide encoding same.

8. Use of FAM19a5 or a homologous protein thereof or a polynucleotide encoding the same as defined in claim 1 as a target for the manufacture of a formulation for diagnosing diseases including depression, cognitive disorders and neuroinflammation.

9. The use according to claim 8, wherein said preparation is a preparation for measuring the mRNA expression level or the protein expression level of said FAM19A5 or its homologous protein gene.

10. The use of claim 9, wherein said agent comprises an oligonucleotide having a sequence complementary to said mRNA, a primer or nucleic acid probe that specifically binds to said mRNA, or an antibody specific for FAM19a5 protein.

Technical Field

The invention relates to the field of genetic engineering, in particular to FAM19A5 protein with multiple functions, a polynucleotide encoding the protein, a genetic engineering vector containing the polynucleotide, a corresponding pharmaceutical composition, and medical application of the FAM19A5 protein, the polynucleotide and the pharmaceutical composition in diagnosing, preventing or treating diseases such as depression, cognitive disorder, neuroinflammation and the like.

Background

Depression is a common mental disorder and is also the leading cause of disability and suicide, with a prevalence of about 17%. Depression contains several persistent symptoms including depressed mood, anhedonia, irritability, and mental retardation. In addition, depressed patients also experience hippocampal-dependent cognitive changes. Depression is caused by both genetic and environmental risk factors. Among them, environmental factors such as stress play an important role in the occurrence and development of depression, resulting in changes in gene expression and neural circuit function, and finally in behavior.

Cytokines are small soluble proteins synthesized and secreted by various cells of the body, have various physiological activities and participate in pathological reactions. Cytokines provide the cells with the ability to communicate with each other and mediate complex multicellular behaviors. Numerous studies have shown that many cytokines play important roles in complex central nervous system functions. Inflammatory cytokine levels are elevated in one third of depressed patients, and clinical depression occurs in up to 40% of patients receiving cytokine therapy. In some prospective human studies, some proinflammatory cytokines, such as interleukin-1 β (interleukin-1 β, IL-1 β), interleukin-6 (interleukin-6, IL-6), and Tumor Necrosis Factor (TNF), are associated with cognitive impairment. Changes in the expression of some chemokines, such as CXCL12, CCL2 and CX3CL1, and their respective receptors, are also increasingly found to be involved in the pathogenesis of central nervous system diseases such as depression, cognitive disorders, neuroinflammation, and the like.

The detection methods of gene expression and protein expression of the encoded product thereof include detection methods such as reverse transcription-polymerase chain reaction, western blotting, ELISA, FACS, immunofluorescence, immunohistochemistry, immunocytochemistry, and the like. Can be applied to experimental research and detection of gene expression of cells and tissues and protein expression of coded products thereof in clinical physiological and pathological diseases (such as depression, cognitive disorder, neuroinflammation and immunoregulation).

The gene with important physiological and pathological significance and the protein of the coded product thereof can be used as a drug target, develop a compound, an antibody, a polypeptide drug or a genetic engineering drug and a commercialized reagent which target the molecule and the interactive molecule thereof, and are applied to the research of pathogenesis, the research of disease markers, clinical detection and drug treatment.

Disclosure of Invention

The invention discloses a cell factor with chemotactic activity, and the research of the invention discovers that FAM19A5 protein, FAM19A5 secretory protein, polynucleotide for coding FAM19A5, a genetic engineering vector containing the polynucleotide and a corresponding pharmaceutical composition have the functions of regulating depression-related mood, cognition-related behavior and neuroinflammation, and can be applied to diagnosis, prevention or treatment of depression, cognitive disorder, neuroinflammation and the like.

In one aspect, the invention provides a protein capable of modulating depression, cognitive disorders, and neuroinflammation. The protein is:

(1) has the sequence shown in SEQ ID No: 2 or SEQ ID No: 3; or

(2) A protein having at least 80% amino acid sequence homology with the protein of (1), and having the same or similar biological functions as the protein of (1).

SEQ ID No: 2, the full-length amino acid sequence of the human FAM19A5 protein, SEQ ID No: 3 shows that human FAM19A5 secretes protein, which retains 89 amino acids at C-terminal of full-length FAM19A5 protein and has the same activity. Has a sequence similar to SEQ ID No: 2 or SEQ ID No: 3, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, particularly preferably at least 98%, and even more particularly preferably at least 99% homologous (sequence match) to the amino acid sequence of SEQ ID No: 2 or SEQ ID No: 3, and proteins with the same or similar biological functions also belong to the category of the proteins for regulating depression, cognitive impairment and neuroinflammation.

In another aspect, the present invention provides a polynucleotide encoding the protein of the present invention, which is capable of modulating depression, cognitive impairment and neuroinflammation. The polynucleotide is:

(i) encoding SEQ ID No: 2 or SEQ ID No: 3; or

(ii) (ii) a polynucleotide having at least 80% sequence homology to the polynucleotide of (i) which encodes a protein having the same or similar biological function as the protein encoded by the polynucleotide of (i).

SEQ ID No:1, given a polynucleotide sequence encoding the full-length FAM19a5 protein, one skilled in the art would appreciate, based on codon degeneracy, that the gene encoding the FAM19a5 protein is not limited to SEQ ID No: 1. in addition, the polynucleotide provided by the present invention may be added with non-coding sequences, such as introns, non-coding sequences 5 'or 3' to the coding sequence, tag sequences, etc., based on the protein coding sequence. Accordingly, the polynucleotides capable of modulating depression, cognitive disorders and neuroinflammation according to the present invention further comprise polynucleotides having at least 80%, preferably at least 85%, more preferably at least 90% homology to the polynucleotide sequence encoding FAM19a5 protein, and the polynucleotides encoding proteins having the same or similar biological function as FAM19a 5. The polynucleotide sequences of the present invention are preferably provided in "isolated" form. By "isolated" form, it is meant that the protein associated with it has not only been separated from the protein in the cell, but has also been separated from the sequences that flank it in nature.

In another aspect, the present invention provides a genetically engineered vector comprising a polynucleotide encoding a protein of the present invention. The genetic engineering vector can be a common vector, an expression vector, a plasmid vector, a virus vector and the like. Preferably, the genetic engineering vector of the invention is an adeno-associated virus genetic engineering vector, and one genetic engineering vector provided in the embodiment of the invention is adeno-associated virus-FAM19A5(AAV9-hSyn-hFAM19A5) containing a neuron-specific promoter.

In another aspect, the invention provides a method for implementing the protein of the invention and the polynucleotide encoding the protein, a genetic engineering vector containing the polynucleotide, and a corresponding pharmaceutical composition.

In another aspect, the invention provides a pharmaceutical composition comprising a protein polypeptide, polynucleotide and/or genetic engineering vector of the invention, and one or more pharmaceutically acceptable salts or pharmaceutically acceptable carriers or excipients.

In another aspect, the invention provides the use of the protein or polynucleotide of the invention, a genetically engineered vector comprising the polynucleotide, and a corresponding pharmaceutical composition in the preparation of a medicament for the prevention and/or treatment of diseases, wherein the diseases include depression, cognitive disorders, neuroinflammation.

In another aspect, the present invention provides a method for detecting whether the expression level of a protein or polynucleotide of the present invention in a test sample is altered. These methods include, but are not limited to, reverse transcription-polymerase chain reaction, western blot, immunomicrosphere, immunohistochemical detection, immunocytochemical detection.

In another aspect, the invention provides the use of a protein of the invention or a polypeptide fragment thereof, or a polynucleotide encoding the protein or polypeptide, in the preparation of a commercial agent for the study of diseases, including depression, cognitive disorders, neuroinflammation. The commercial agent may comprise a protein of the invention or a polypeptide fragment thereof or a polynucleotide encoding the same, or may comprise a compound, an antibody, a polypeptide or an oligonucleotide targeting a protein of the invention or a polypeptide fragment thereof or a polynucleotide encoding the same.

In another aspect, the invention provides the use of the protein polypeptides or polynucleotides of the invention as targets for the development of compounds, antibodies, polypeptide pharmaceuticals and commercial agents. For example, an agent for measuring the mRNA expression level of FAM19a5 gene or the protein expression amount thereof can be used for diagnosing depression, cognitive disorders, and neuroinflammation. Such a preparation may include, for example, an oligonucleotide having a sequence complementary to FAM19a5 mRNA, a primer or nucleic acid probe that specifically binds to FAM19a5 mRNA, and an antibody specific for FAM19a5 protein.

The invention proves that the FAM19A5 protein has an important role in maintaining the regulation of cognition and depression-related mood for the first time, thereby obtaining a prevention and treatment means for depression and cognitive disorder-related diseases. FAM19a5 may also be used as a biomarker for diagnosing depression and cognitive disorder related diseases.

Drawings

FIG. 1 shows a histogram of flow cytometry detection of FAM19A5 recombinant protein at a known concentration of 0-32000pg/mL in example 1.

FIG. 2 shows a standard curve of a double antibody sandwich microsphere system for detecting the amount of FAM19A5 in a tissue sample according to example 1. Tissue lysine: tissue lysate.

FIG. 3 shows a standard curve of a double antibody sandwich microsphere system for detecting the amount of FAM19A5 in a plasma sample in example 1. Plasma: plasma.

FIG. 4 shows the expression profile of FAM19A5 in each tissue of mice as measured by Real-time PCR in example 2. The results show that: FAM19a5 was highly expressed in mouse brain. Relative mRNA level: relative mRNA expression levels.

FIG. 5 shows the expression profile of FAM19A5 in each mouse tissue in the double-antibody sandwich microsphere detection of example 2. The results show that: FAM19a5 was highly expressed in mouse brain. Concentration: the protein concentration.

Figure 6 shows the double-antibody sandwich microspheres of example 2 to detect the concentration of FAM19a5 in plasma and cerebrospinal fluid of mice. The results show that: FAM19a5 was present at approximately the same concentration in plasma and cerebrospinal fluid of mice. Concentration: protein concentration; plasma: plasma; CSF: cerebronasal fluid, cerebrospinal fluid.

FIG. 7 shows the Real-time PCR detection of FAM19A5 expression at various developmental stages in mice in example 3. The results show that: at the mRNA level, FAM19A5 expression increased slightly starting at 12.5 days from mouse embryonic stage, peaked at 1 day after birth and then decreased at 7 days after birth. Suggesting that the expression of FAM19a5 may be involved in brain development. E12.5: embryonic day 12.5, Embryonic stage 12.5 days; p1: postnatal day 1, the day after birth; 8W: 8Week, eight weeks. Relative mRNA level: relative mRNA expression levels.

FIG. 8 shows the double-antibody sandwich microspheres of example 3 to detect the expression of FAM19A5 at various developmental stages in mice. The results show that: at the protein level, FAM19a5 expression rose slightly starting at embryonic stage 12.5 days in mice, then continued to rise at embryonic stage 16.5 days and postnatal day 1, peaking at 7 days postnatal and then declining at 8 weeks. Suggesting that the expression of FAM19a5 may be involved in brain development. E12.5: embryonic day 12.5, Embryonic stage 12.5 days; p1: postnatal day 1, the day after birth; 8W: 8Week, eight weeks. Concentration: the protein concentration.

FIG. 9 shows the Real-time PCR detection of FAM19A5 expression in different brain regions of mice in example 4. The results show that FAM19a5 is highly expressed in the hippocampus of mouse brain tissue, and exhibits moderate levels of expression in amygdala, thalamus, cerebral cortex, hypothalamus and cerebellum. Hippocampus: hippocampal region; amygdala: almond kernel; thalamus: the thalamus; cerebral core: cerebral cortex; hypothalamus: the hypothalamus; cerebellum: the cerebellum. Relative mRNA level: relative mRNA expression levels.

FIG. 10 shows the double-antibody sandwich microsphere assay of example 4 for the expression of FAM19A5 in different brain regions of mice. The results show that FAM19a5 is highly expressed in the hippocampus of mouse brain tissue, and exhibits moderate levels of expression in amygdala, thalamus, cerebral cortex, hypothalamus and cerebellum. Hippocampus: hippocampal region; amygdala: almond kernel; thalamus: the thalamus; cerebral core: cerebral cortex; hypothalamus: the hypothalamus; cerebellum: the cerebellum. Concentration: the protein concentration.

FIG. 11 shows immunohistochemical detection of FAM19A5 expression in different brain regions of mice in example 4. The results show that FAM19A5 is highly expressed in CA1 region of hippocampus of mouse brain tissue, especially in centrum neurons, and exhibits moderate expression in amygdala, thalamus, cerebral cortex, hypothalamus and cerebellum. Scale bar 50 μm.

FIG. 12 shows example 5 immunofluorescence detection of co-expression of FAM19A5 with the neuron-specific marker NeuN on hippocampus, cortex, and primary cultured neurons of adult mice. The results show that: the neurons express FAM19a 5. Neuron: a neuron; hippocampus: hippocampal region; cerebral core: cerebral cortex; scale 25 μm.

FIG. 13 shows example 5 immunofluorescence detection of co-expression of FAM19A5 with neural stem cell specific marker Nestin on hippocampus and primary cultured neural stem cells of embryonic mice. The results show that: neural stem cells express FAM19a 5. Hippocampus: hippocampal region; neural stem cell: a neural stem cell; the upper scale is 100 μm and the lower scale is 25 μm.

FIG. 14 shows example 5 immunofluorescence detecting co-expression of FAM19A5 with the oligodendrocyte-specific marker Olig 2. The results show that: oligodendrocytes express FAM19a 5. Scale 25 μm.

FIG. 15 shows immunofluorescence detection of co-expression of FAM19A5 with glutamatergic neuron specific markers VGLUT2 in example 5. The results show that: glutamatergic neurons express FAM19a 5. Scale bar 10 μm.

FIG. 16 shows the Real-time PCR assay of example 5 for FAM19A5 expression in HEK293T, neurons, astrocytes and neural stem cells. The results showed that neurons and neural stem cells expressed FAM19a 5. Neuron: a neuron; astrocyte: astrocytes; neural stem cell: neural stem cells. Relative mRNA level: relative mRNA expression levels.

FIG. 17 shows the double-antibody sandwich microspheres of example 5 to detect the expression of FAM19A5 in HEK293T, neurons, astrocytes and neural stem cell culture supernatants. The results showed that neurons and neural stem cells expressed and secreted FAM19a 5. Neuron: a neuron; astrocyte: astrocytes; neural stem cell: neural stem cells. Concentration: the protein concentration.

FIG. 18 shows detection of Fam19a5 knockdown (Fam19 a5) in the open field experiment of example 6^-/-) Distance of movement of mice and littermate Wild Type (WT) mice within 30 minutes of measurement. As can be seen, Fam19a5^-/-Mice exhibited a significant increase in exercise capacity. Distance: a distance; min: and (3) minutes.

FIG. 19 shows the detection of Fam19a5 in the open field experiment of example 6^-/-Time (%) occupied in the central zone within the first 5 minutes of measurement for the mouse and the WT mouse. As can be seen, Fam19a5^-/-Mice showed a decrease in time to enter the central zone. Note that Fam19a5^-/-The mice exhibited an increase in depression-like behavior. Time in center: the time in the central zone.

FIG. 20 shows the detection of Fam19a5 in the open field experiment of example 6^-/-The movement speed of the mice and WT mice within 30 minutes of the measurement. As can be seen, Fam19a5^-/-Mice show increased locomotor speed. Move speed: the speed of movement.

FIG. 21 shows the sucrose preference assay of example 6 for detecting Fam19a5^-/-Preference of mice and WT mice for sucrose solutions. As can be seen, Fam19a5^-/-The intake of sucrose solution in mice was significantly reduced, showing anhedonia. Note that Fam19a5^-/-The mice exhibited an increase in depression-like behavior. The Sucrose prediction: sucrose preference.

FIG. 22 shows the test results of forced swimming in example 6Fam19a5 test^-/-The despair behavior of mice and WT mice under acute stress. As can be seen, Fam19a5^-/-The immobility time of the mice is increased, and despair behaviors are increased. Note that Fam19a5^-/-The mice exhibited an increase in depression-like behavior. Mobility: and (4) standing still.

FIG. 23 shows the detection of Fam19a5 in the tail suspension experiment of example 6^-/-The despair behavior of mice and WT mice under acute stress. As can be seen, Fam19a5^-/-The immobility time of the mice is increased, and despair behaviors are increased. Note that Fam19a5^-/-The mice exhibited an increase in depression-like behavior. Mobility: and (4) standing still.

FIG. 24 shows the test Fam19a5 of the water maze experiment of example 7^-/-Mice were looking for the latency of the underwater platform during the training phase. As can be seen, Fam19a5^-/-Mice seek increased latency to the platform. Note that Fam19a5^-/-Mice exhibit spatial cognitive impairment. Escape latency: escape latency; flag: a flag; day: day; time: time; sec: and second.

FIG. 25 shows the test Fam19a5 of the water maze experiment of example 7^-/-Mice were looking for a trajectory map of the platform during the test period. As can be seen, Fam19a5^-/-Mice had less time in the target quadrant where the platform was located. Note that Fam19a5^-/-Mice exhibit spatial cognitive impairment. KO: fam19a5 knockout mice.

FIG. 26 shows the test Fam19a5 of the water maze experiment of example 7^-/-Mice were in the quadrant of the platform during the test period. As can be seen, Fam19a5^-/-Mice had less time in the target quadrant where the platform was located. Note that Fam19a5^-/-Mice exhibit spatial cognitive impairment. TQ, target quadrant (quadrant where the platform is during training); AR, adjacentquart rectangle right, adjacent the right quadrant; OQ, opposite quadrant, contralateral quadrant; AL, adjacentqjacent quadrant left, adjacent to the left quadrant. Time in quadrants: the time occupied in the quadrant.

FIG. 27 shows the test Fam19a5 of the water maze experiment of example 7^-/-Number of times the mouse crossed the position of the platform during the test period. As can be seen, Fam19a5^-/-Where the mouse passes through the platformThe number of positions is reduced. Note that Fam19a5^-/-Mice exhibit spatial cognitive impairment. Number of target cross: number of passes over the location of the platform.

Figure 28 shows the dual antibody sandwich microsphere assay of example 8 expression of FAM19a5 in plasma of mice in the Chronic Restraint Stress (CRS) model and the Chronic Forced Swim Stress (CFSS) model. The results show that: FAM19a5 was down-regulated in plasma from chronic stress model mice. Control: comparison; concentration: the protein concentration. Plasma: plasma.

FIG. 29 shows the Real-time PCR assay of example 8 for the expression of FAM19A5 in the hippocampus of mice in the chronic immobilization stress (CRS) model and the Chronic Forced Swim Stress (CFSS) model. The results show that: FAM19a5 was down-regulated in plasma from chronic stress model mice. Hippocampus: hippocampal region; control: comparison; relative mRNA level: relative mRNA expression levels.

Figure 30 shows the expression of FAM19a5 in the hippocampus of the double antibody sandwich microsphere assay of example 8 in mice of the chronic immobilization stress (CRS) model and the Chronic Forced Swimming Stress (CFSS) model. The results show that: FAM19a5 was down-regulated in plasma from chronic stress model mice. Hippocampus: hippocampal region; control: comparison; concentration: the protein concentration.

Figure 31 shows the expression of FAM19a5 in plasma of chronic social frustration stress (CSDS) model mice as measured by the double-antibody sandwich microsphere of example 8. The results show that: FAM19a5 was down-regulated in plasma from chronic stress model mice. Plasma: plasma; control: comparison; resilient: a recovery type; susceptable: and (4) a susceptible type.

Figure 32 shows the expression of FAM19a5 in the hippocampus of a chronic social frustration stress (CSDS) model mouse as measured by the double-antibody sandwich microsphere of example 8. The results show that: FAM19a5 was down-regulated in plasma from chronic stress model mice. Hippocampus: hippocampal region; control: comparison; concentration: the protein concentration.

FIG. 33 shows example 9 open field experiments to test the number of times a chronic restraint stress-induced depression model mouse overexpressing AAV-Null/AAV-FAM19A5 in the hippocampus entered the central zone within the first 5 minutes of measurement. It can be seen that chronic restraint stress-induced depression model mice overexpressing AAV-FAM19A5 in the hippocampus showed an increased number of passages into the central zone. It was suggested that overexpression of AAV-FAM19A5 in the hippocampus reversed depressive-like behavior in chronic restraint stress-induced depressive model mice. Entries into center: number of entries into the central region.

FIG. 34 shows the open field experiment of example 9 to examine the time (%) at which chronic restraint stress-induced depression model mice overexpressing AAV-Null/AAV-FAM19A5 in the hippocampus enter the central zone within the first 5 minutes of measurement. It can be seen that chronic restraint stress-induced depression model mice overexpressing AAV-FAM19A5 in the hippocampus exhibited increased time to entry into the central zone. It was suggested that overexpression of AAV-FAM19A5 in the hippocampus reversed depressive-like behavior in chronic restraint stress-induced depressive model mice. Time in center: the time in the central zone.

FIG. 35 shows the tail suspension experiment of example 9 to test the despair behavior of chronic restraint stress-induced depression model mice overexpressing AAV-Null/AAV-FAM19A5 in the hippocampus against acute stress. It can be seen that chronic restraint stress-induced depression model mice overexpressing AAV-FAM19a5 in the hippocampus exhibited less immobility time, as evidenced by decreased despair behavior. It was suggested that overexpression of AAV-FAM19A5 in the hippocampus reversed depressive-like behavior in chronic restraint stress-induced depressive model mice. Mobility: and (4) standing still.

FIG. 36 shows immunofluorescence staining of hippocampal tissue following in situ injection of AAV-Null/AAV-FAM19A5 in hippocampal CA1 region of example 9. Since the AAV-FAM19A5 virus vector has a Flag tag at the C-terminus, the expression of Flag can be detected to reflect the expression of FAM19A 5. The results show that: in situ injection of AAV-FAM19A5 can over-express FAM19A5 in the hippocampal CA1 region. Scale bar 50 μm.

FIG. 37 shows that after in situ injection of AAV-Null/AAV-FAM19A5 into the hippocampal CA1 region of example 9, double antibody sandwich microspheres tested FAM19A5 expression in hippocampal and cortical tissues. The results show that: the expression of FAM19A5 in hippocampus and cortex of mice injected with AAV-FAM19A5 in situ was significantly higher than in control group. Concentration: the protein concentration. Hippocampus: hippocampal region; cerebral core: the cerebral cortex.

FIG. 38 shows flow cytometry analysis of WT and Fam19a5 in example 10^-/-The proportion of neutrophils, monocytes/macrophages, T lymphocytes and B lymphocytes in the peripheral blood of the mice. The results show that: fam19a5 compared to WT mice^-/-CD4 in peripheral blood of mice⁺The proportion of T lymphocytes is obviously increased, and the proportion of B lymphocytes is obviously decreased. Neutrophil: a neutrophil granulocytes; monocyte: (ii) a monocyte; blood: peripheral blood. B cells: b lymphocytes; t cells: t lymphocytes.

FIG. 39 shows flow cytometry analysis of WT and Fam19a5 in example 10^-/-The proportion of neutrophils, monocytes/macrophages, T lymphocytes and B lymphocytes in the mouse brain. The results show that: fam19a5 compared to WT mice^-/-The monocyte/macrophage ratio in the mouse brain was significantly up-regulated. Neutrophil: a neutrophil granulocytes; monocyte/macrocage: monocytes/macrophages; b cells: b lymphocytes; t cells: t lymphocytes. Brain: the brain.

FIG. 40 shows flow cytometry analysis of Fam19a5 in example 11^-/-The content of FAM19A5 in the plasma and cerebrospinal fluid of the mice is obtained after the mice are subjected to intravenous injection or intraperitoneal injection of FAM19A5 eukaryotic protein. The results show that: FAM19A5 protein was detected in the plasma of mice either after tail vein injection or intraperitoneal injection of protein, whereas FAM19A5 protein was detected in the cerebrospinal fluid of mice only after 5 minutes of tail vein injection. i.v.: tail vein injection; i.p.: and (5) carrying out intraperitoneal injection. BL: plasma; CSF: and (4) cerebrospinal fluid.

FIG. 41 shows immunofluorescence detection of WT and Fam19a5 in example 12^-/-Staining of neuron-specific marker NeuN and microglia-specific marker Iba-1 in mouse cortex and hippocampus. The results show that: fam19a5^-/-The positive staining of Iba-1 was greater in the mouse cortex and hippocampus. Hippocampus: hippocampal region; cerebral core: the cerebral cortex. Scale bar 50 μm.

FIG. 42 shows the inflammatory phenotype of microglia line BV2 after addition of FAM19A5 eukaryotic protein and LPS as measured by Real-time PCR in example 13. The results show that: FAM19a5 can inhibit the inflammatory phenotype of the microglia line BV 2.

FIG. 43 shows the proliferation and self-renewal of neural stem cells after 24 hours of stimulation with various concentrations of FAM19A5 recombinant protein, as measured in example 14 CCK-8. The results show that: FAM19a5 can inhibit proliferation and self-renewal of neural stem cells.

FIG. 44 shows the detection of WT and Fam19a5 in example 14CCK-8^-/-Proliferation and self-renewal of mouse neural stem cells, and proliferation and self-renewal of neural stem cells stimulated by administration of FAM19A5 recombinant protein. The results show that: fam19a5^-/-The proliferation capacity of the mouse primary neural stem cells is enhanced, the number and the size of second-generation neurospheres are increased, and the FAM19A5 recombinant protein can reverse the proliferation and self-renewal capacity of the mouse primary neural stem cells.

Detailed Description

The present invention is illustrated in more detail below by means of some examples. It should be understood, however, that these descriptions and the examples listed below are not intended to limit the scope of the present invention.

The FAM19A5 adeno-associated virus expression system is constructed, FAM19A5 protein is over-expressed in CA1 region of mouse hippocampus in a stereotactic injection mode, and the FAM19A5 adeno-associated virus expression system plays a role in treating chronic stress-induced depression, and is the first discovery internationally through experiments.

Experiments prove that the FAM19A5 protein-expressing adeno-associated virus has a therapeutic effect on chronic stress-induced depression, and the FAM19A5 protein-encoding polynucleotide is derived from a sequence encoding human FAM19A5 protein.

According to a specific embodiment of the present invention, FAM19a5 is specifically highly expressed in the brain of a vertebrate. The mouse is subjected to stereotactic injection of AAV-FAM19A5 to relieve the depression phenotype of the mouse induced by chronic stress, so that FAM19A5 is known to be an important protective factor of brain tissue. Thus, the present invention demonstrates for the first time the regulatory role of FAM19a5 in depression.

Accordingly, the present invention provides a pharmaceutical composition for modulating depression, the composition comprising AAV-FAM19a5 or other vectors expressing FAM19a5 or FAM19a5 protein polypeptide.

According to another embodiment of the present invention, FAM19a5 is specifically highly expressed in the brain, particularly in the hippocampus. Neurons, neural stem cells and oligodendrocytes may express FAM19a 5. Constructing Fam19a5 knockout mice demonstrates that the absence of Fam19a5 results in mice exhibiting increased depressive-like behavior and cognitive impairment. Expression of FAM19a5 was down-regulated in hippocampus and plasma of chronic stress-induced depression model mice. Through stereotactic injection of AAV-FAM19A5 containing neuron specific promoter into CA1 region of hippocampus, FAM19A5 is overexpressed in hippocampus, so that chronic stress-induced depression-like behavior can be relieved, and FAM19A5 protein is known as a protective factor for mood regulation related to spatial cognition and depression. Therefore, the FAM19A5 protein is proved to have an important role in maintaining cognitive and depression-related mood regulation for the first time or to be used as a therapeutic means for depression and cognitive disorder-related diseases.

Accordingly, the present invention provides a pharmaceutical composition for participating in the regulation of mood associated with the maintenance of cognition and depression, which comprises FAM19a5 protein or a polynucleotide encoding FAM19a5 that can function as a protein involved in the regulation of mood associated with the maintenance of cognition and depression, a genetically engineered vector comprising the polynucleotide, and a corresponding pharmaceutical composition. The preferred genetic engineering vector is adeno-associated virus AAV9-hSyn-hFAM19A5 containing neuron specific promoter.

The composition for maintaining cognitive and depression-related mood regulation of the present invention may comprise native or recombinant FAM19a5, a FAM19a5 protein having substantially equivalent physiological activity thereto, a transgenic neuronal cell overexpressing said native or recombinant FAM19a5, or a FAM19a5 inhibitor. The protein having substantially equivalent physiological activity comprises natural/recombinant FAM19a5, functional equivalents and functional derivatives thereof.

The detection of the behavior related to the depression of the mouse is a widely used mouse open field experiment, a sucrose preference experiment, a forced swimming experiment and a tail suspension experiment.

The mouse cognition-related behavior detection is a widely used mouse water maze experiment.

The depression model induced by Chronic stress of mice is considered to better reflect depression of human beings, and comprises Chronic Restraint Stress (CRS), Chronic Forced Swimming Stress (CFSS) and Chronic social frustration stress (CSDS).

The term "functional equivalents" refers to amino acid sequence variants and over-expression vectors in which some or all of the amino acids of the native protein have been substituted or some of the amino acids have been deleted or added, and which have substantially equivalent biological activity to native FAM19a 5.

The term "functional derivative" refers to a modified protein that enhances or decreases the physical and chemical properties of the FAM19a5 protein and has substantially equivalent biological activity to native FAM19a 5.

The FAM19a5 inhibitor may be any one of an antisense oligonucleotide, siRNA, shRNA, miRNA or a vector comprising the same, or a receptor sequence, or an antibody. The antisense strand of the invention can be the complementary sequence of the sequence shown in SEQ ID NO. 1. It is known to those of ordinary skill in the art that antisense strands or portions thereof (antisense oligonucleotides) can be used to inhibit expression of FAM19a5 protein in cells.

Furthermore, according to another embodiment of the present invention, FAM19a5 is down-regulated as depression develops in a mouse stress-induced depression model. The absence of FAM19a5 caused spontaneous depression-like behavior and cognitive impairment in mice, and therefore FAM19a5 could be expected to have an important role in cognitive and depression-related mood regulation in vivo in the long run. Thus, FAM19a5 is useful as a biomarker for diagnosing depression and cognitive disorder-related diseases.

The term "stress" as used in this specification includes the non-specific response of an individual by various stressors (stressors). For example, sympathetic nerve excitation, increased secretion of pituitary and adrenal cortical hormones, increased blood sugar, increased blood pressure, increased heart rate and respiration, emotional response and self-defense response, response to stress, and the like, but the stress is not limited thereto.

The term "depression" means that symptoms include depressed mood, depression, and even pessimistic boredom, with suicide attempts or behaviors; even the occurrence of stupor; in some cases, there is significant anxiety and motor agitation; the serious patient may have a mood disorder disease with psychotic symptoms such as hallucinations and delusions, but the disease is not limited to any particular disease category.

The term "cognitive disorder-related disease" refers to a pathological process in which a higher intelligent processing process of the brain related to learning, memory and thinking judgment is abnormal, thereby causing severe learning and memory disorder and accompanied with changes such as aphasia, misuse, agnosia, or disability, but the disease is not limited to any specific disease category.

The term "diagnosis" refers to the determination of a pathological state. For the purposes of the present invention, the diagnosis is to determine FAM19a5 expression as a diagnostic marker for depression and cognitive disorders to confirm the onset, progression, and remission of depression and cognitive disorders.

The term "diagnostic marker" refers to a substance of a cell capable of diagnosing depression and cognitive impairment from common cells, which includes organic biomolecules such as polypeptides or nucleic acids (e.g., mRNA), lipids, glycolipids, glycoproteins, and sugars (monosaccharides, disaccharides, oligosaccharides, etc.), which are increased or decreased in depression and cognitive impairment cells compared to normal cells. The diagnostic marker for depression and cognitive impairment provided by the present invention may be a protein expressed by FAM19a5 gene.

The composition for diagnosing depression and cognitive impairment of the present invention comprises an agent for measuring the mRNA expression level of FAM19a5 gene or the protein expression amount thereof. Such a preparation may include, for example, an oligonucleotide having a sequence complementary to FAM19a5 mRNA, a primer or nucleic acid probe that specifically binds to FAM19a5 mRNA, and an antibody specific for FAM19a5 protein.

Furthermore, according to another embodiment of the present invention, the deletion of FAM19a5 results in a change in immune cell composition in peripheral blood and brain tissue of mice compared to wild type mice. Changes in immune cell composition in brain tissue can affect neuroinflammation.

The pharmaceutical composition for diagnosing depression, cognitive impairment, and neuroinflammation of the present invention may be in the form of a kit contained therein. The kit may comprise the primer, probe or antibody for measuring the expression level of FAM19a5 gene or the amount of protein.

When the kit is used in a PCR amplification process, reagents necessary for PCR amplification may optionally include, for example, a buffer, a DNA polymerase cofactor, and dNTPs. When the kit is used in an immunoassay, it may optionally comprise a secondary antibody and a labeled substrate. Further, the kits of the present invention may be made in a plurality of separate packages or compartments (compartments) capable of containing the reagent compounds described above.

Methods for measuring the expression level of a gene or the amount of a protein expressed are well known techniques, which include known processes for isolating mRNA or protein from a biological sample.

The biological sample refers to a sample collected from organisms having different gene or protein expression levels when the degree of onset or progression of depression, cognitive impairment, and neuroinflammation is compared with that of a normal control group. Examples of the sample may include, but are not limited to, tissue, cells, blood, serum, plasma, saliva, and urine.

When measuring the expression level of the gene, it is preferable to measure the level of mRNA. As a method for measuring the level of mRNA, RT-PCR, real-time PCR, RNase protection assay, Northern blotting, DNA chip, etc. can be used, but not limited thereto.

When measuring the protein level, antibodies may be used. At this time, FAM19a5 protein and its specific antibody in the biological sample may form a conjugate (i.e., an antigen-antibody complex). The amount of the antigen-antibody complex formed can be quantitatively determined by the magnitude of the signal generated by the detection label. The detectable label may be selected from the group consisting of an enzyme, a fluorescent ligand, a luminescent substance, a microparticle, a redox molecule, and a radioisotope, but is not limited thereto. Analytical methods for determining protein levels include, but are not limited to, Western blotting, ELISA, radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation analysis, complement fixation analysis, FACS, protein chips, and the like.

Thus, by using the detection method, the present invention can determine the amount of mRNA or protein expressed in a control group, and the amount of mRNA or protein expressed in a patient suspected of suffering from depression, cognitive disorders, and neuroinflammation. Then, the results can be compared with each other to diagnose the onset, progression, and the like of depression, cognitive impairment, neuroinflammation.

Further, in the method for diagnosing depression, cognitive impairment, and neuroinflammatory disorders of the present invention, when the expression level of FAM19a5 gene of the present invention or the amount of the expressed protein is differentiated compared to the normal control group sample, the presence of depression, cognitive impairment, and neuroinflammatory disorders can be judged.

The present invention also provides a method for screening a prophylactic or therapeutic drug for depression, cognitive disorders and neuroinflammatory diseases, the method comprising: contacting the FAM19a5 gene with a candidate substance in vitro, and determining whether the candidate substance can promote or inhibit expression of the gene; alternatively, FAM19a5 protein is contacted with a candidate substance in vitro, and it is determined whether the candidate substance increases or inhibits the function or activity of the protein.

According to the screening method of the present invention, first, a candidate substance to be analyzed is contacted with cells of depression and cognitive impairment diseases containing the gene or protein.

According to conventional selection methods, the candidate substance may comprise: substances capable of promoting or inhibiting transcription into mRNA and translation into protein in the FAM19a5 gene sequence, substances presumed to have the possibility of promoting or inhibiting the function or activity of FAM19a5 protein for medical use, or individual nucleic acids, proteins, peptides, other extracts, natural products, compounds, and the like selected at random.

Then, the amount of gene expression, the amount of protein or the activity of protein is measured in the cells treated with the candidate substance. In the measured results, when an increase or decrease in the amount of gene expression, the amount of protein or the activity of protein is detected, the candidate substance can be judged as a substance capable of treating or preventing depression and cognitive impairment diseases.

As described above, the amount of gene expression, the amount of protein or the activity of protein can be measured by various methods known in the art, such as RT-PCR, real-time polymerase chain reaction, Western blotting, Northern blotting, enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), radioimmunodiffusion, immunoprecipitation, etc., but not limited thereto.

By the screening method of the present invention, a candidate substance exhibiting a property capable of promoting gene expression or promoting protein function, and conversely a candidate substance exhibiting a property capable of suppressing gene expression or suppressing protein function, can be obtained.

Therapeutic agent candidates for depression, cognitive disorders and neuroinflammatory disorders may play a leading role of the compound in the development of later therapeutic agents for depression, cognitive disorders and neuroinflammatory disorders. When the structure of the leading compound is modified and optimized to promote or inhibit the function of FAM19a5 gene or its expressed protein, a novel therapeutic agent for depression, cognitive disorders and neuroinflammatory diseases can be developed.

The invention also provides application of the FAM19A 5-encoding polynucleotide or FAM19A5 secretory protein in preparation of commercial reagents for researching depression, cognitive disorder and neuroinflammation diseases.

In the present invention, the contents related to genetic engineering techniques can be clearly understood from those disclosed in Sambrook, et al, molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001) and Frederick M.Ausubel et al, Current protocols in molecular biology volume1,2,3, John Wiley & Sons, Inc. (1994)).

Example 1 establishment of FAM19A5 double antibody sandwich microsphere detection System

The method comprises the following steps:

mu.L of aldehyde/sulfuric acid latex microspheres and 20. mu.L of rabbit anti-FAM 19A5 polyclonal antibody (2.5mg/mL) were added to a final volume of 200. mu.L of MES Buffer (0.025mol/L, pH 6.0) and spun slowly overnight at 4 ℃. FAM19A5 rabbit polyclonal antibody coated microspheres were treated according to Latex Bead Protein Coupling Protocols (Thermo Fisher Scientific). The procedure for microsphere detection was as follows:

microsphere pretreatment: mu.L of microspheres per sample was added 1. mu.L of blocking solution (10% BSA in PBS or 1000-fold diluted Fam19a5)^-/-Mouse plasma) the required amounts of microspheres and blocking solution were calculated, mixed and incubated at room temperature for 30 minutes.

Sample pretreatment: the supernatant was aspirated after centrifugation of the homogenate at 3000g/5 min, quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), and diluted to a final concentration of 1mg/mL, based on the Protein concentration of the homogenate, in PBS, in a total volume of 50. mu.L. Mouse cerebrospinal fluid and plasma are centrifuged at 3000g/5 min, and the supernatant is aspirated, diluted 2000 times and used, with a total volume of 50. mu.L. The cell culture supernatant was not diluted and the total volume was 50. mu.L. The standard product is 89 amino acid secretion form (SEQ ID No: 3) of FAM19A5 recombinant eukaryotic protein after purification and quantification. The diluent of the standard substance for detecting the tissue homogenate and the cell culture supernatant is 5 percent BSA PBS solution; the dilution of the standard used for detecting cerebrospinal fluid and plasma is Fam19a5 diluted 2000 times^-/-Mouse plasma. The concentration of the standard substance after dilution is 0-32000 pg/mL.

Add 50. mu.L of the treated sample to the pretreated 2. mu.L microspheres and mix well before rotary incubation at room temperature for 2 hours. After centrifugation at 3000g/5 min, the supernatant was aspirated.

Resuspend the microspheres in 50. mu.L of detection primary antibody dilution (goat anti-FAM 19A5 polyclonal antibody: 0.2mg/mL, 1/200 diluted to 5% BSA in PBS) and incubate slowly at room temperature with rotation for 1 hour. After centrifugation at 3000g/5 min, the supernatant was aspirated.

Resuspend the microspheres in 50. mu.L of a test secondary antibody dilution (PE-labeled anti-goat IgG: 1/1000 diluted to 5% BSA in PBS) and incubate slowly with spin at room temperature for 0.5 h in the dark. After centrifugation at 3000g/5 min, the supernatant was aspirated.

After resuspending the microspheres in 300. mu.L of PBS, they were examined by FACSVersese flow cytometer. And fitting a standard curve through the concentration of the standard substance and the Geomean value, and calculating the concentration of FAM19A5 of the sample to be detected through the standard curve.

Secondly, the result is:

the concentration of FAM19A5 in the sample was determined by flow cytometry, and a standard curve was prepared by calculation from the geoman value of fluorescence intensity. FIG. 1 shows a histogram of flow cytometry detection of FAM19A5 recombinant protein at known concentrations of 0-32000 pg/mL. FIG. 2 shows the linear coefficient R of the microsphere standard curve for detecting tissue samples²Is 0.9834. FIG. 3 shows the linear coefficient R of the microsphere standard curve for detecting plasma samples²Is 0.9902.

Example 2 expression profiles of FAM19A5 in mouse tissues

The method comprises the following steps:

black male mice (C57BL/6J, mice) were purchased from the laboratory animal department of the department of medicine, Beijing university, 6-8 weeks old. The purchased mice are bred in a mouse cage, proper food and water are supplied in the mouse cage, the temperature is maintained at 20-24 ℃, and the humidity is maintained at 40-70%. These wild-type mice were kept in a 12-hour/12-hour light dark cycle (light was turned on at eight am and light was turned off at eight pm). All experiments were designed to use a minimum number of mice and, depending on the ethics of the animal experiments, anesthesia was performed to minimize the pain of the mice used in the experiments. After cardiac perfusion after mouse anesthesia, organs of the mouse were taken, total RNA was extracted from each corresponding tissue, and complementary dna (cdna) was then prepared by using reverse transcriptase and random hexamers. Subsequently, primers were designed based on the full-length sequence (SEQ ID No: 4) of the gene encoding mouse FAM19A5, and the expression of each organ FAM19A5 was detected by real-time quantitative polymerase chain reaction using the corresponding primers. Real-time PCR amplification was performed for 40 rounds using the sequences of FAM19A5 upstream F (5'-gaccagcagccggcaagatg-3', SEQ ID No: 5), downstream R (5'-cgagcgtccacacaagcagg-3', SEQ ID No: 6), GAPDH upstream F (5'-cggagtcaacggatttggtcgtat-3', SEQ ID No: 7), and downstream R (5'-agccttctccatggtggtgaagac-3', SEQ ID No: 8) as primers. And homogenizing each organ of the mouse, taking the supernatant, and detecting the expression of FAM19A5 in each tissue organ, blood plasma and cerebrospinal fluid by using a double-antibody sandwich microsphere detection system.

Secondly, the result is:

as shown in FIG. 4, Fam19a5 was highly expressed in brain tissue. Further, we verified the protein expression level of FAM19a5 in each mouse tissue, consistent with the mRNA level, and the protein level of FAM19a5 was also significantly high expressed in mouse brain tissue using the microsphere detection system (fig. 5). As shown in fig. 6, the concentration of FAM19a5 was approximately the same in plasma and cerebrospinal fluid.

Example 3 expression of FAM19A5 at different developmental stages in mice

The method comprises the following steps:

mice were managed in the same manner as described above. After anesthesia, the brains were removed from the cranium by cervical amputation and sacrifice of prenatal embryonic day 12.5 (E12.5), day 16.5 (E16.5) and postnatal day 1 (P1), day 7 (P7) and week 8 (8W) mice. And detecting the expression of FAM19A5 in the brain of the mouse at each development stage by using Real-time PCR and a double-antibody sandwich microsphere detection system.

Secondly, the result is:

the results are shown in FIG. 7, where FAM19A5 expression was slightly elevated at the mRNA level starting at 12.5 days from the mouse embryonic stage, with peak expression at 1 day after birth and then decreased at 7 days after birth. As shown in figure 8, at the protein level, FAM19a5 expression increased slightly starting at embryonic stage 12.5 days in mice, then continued to increase at embryonic stage 16.5 days and postnatal day 1, peaking at postnatal day 7 and then decreasing at week 8. Suggesting that the expression of FAM19a5 may be involved in brain development.

Example 4 expression of FAM19A5 in different brain regions of mice

The method comprises the following steps:

mice 6-8 weeks old were managed in the same manner as described above.

Mice for Real-time PCR and double antibody sandwich microsphere detection were sacrificed by cervical amputation after anesthesia and brains were obtained from their cranium. Taking different brain areas (hippocampus, amygdala, thalamus, cerebral cortex, hypothalamus and cerebellum), and detecting the expression of FAM19A5 in each brain area by using Real-time PCR and a double-antibody sandwich microsphere detection system.

Mice for paraffin sectioning:

1) material taking: after anesthetizing the mice with avodine (240mg/kg), the hearts were perfused with cold PBS and 4% Paraformaldehyde (PFA). Mouse brain tissue was fixed in paraformaldehyde solution for 24 hours.

2) And (3) dehydrating: taking out the tissue from the polyformaldehyde solution, washing the tissue with PBS, putting the tissue into a paraffin embedding box, marking the tissue number with a pencil, and sequentially soaking for 24 hours, 12 hours and 2.5 hours respectively in 60 percent, 70 percent and 80 percent ethanol, 1 hour respectively in 100 percent ethanol I and 100 percent ethanol II.

3) And (3) transparency: the tissue was soaked in xylene I and xylene II for 25 minutes, respectively.

4) Wax dipping: the tissue was immersed in paraffin I and paraffin II, which were previously melted by heating, for 45 minutes, respectively.

5) Embedding: and putting the tissues into a module on a paraffin embedding operation table, adding filtered paraffin which is heated and melted in advance, and cooling and forming.

6) Slicing: the prepared wax block was sliced with a thickness of 4 μm using a Leica semi-automatic microtome, and a slide patch pretreated with cations was used. The attached paraffin sections were oven-dried at 60 ℃ for 2 hours.

7) Placing the paraffin slices into dimethylbenzene I and dimethylbenzene II, respectively soaking for 20 minutes, then sequentially placing the paraffin slices into 100% ethanol I, 100% ethanol II, 95% ethanol and 75% ethanol, respectively soaking for 5 minutes, and then placing the paraffin slices into PBS.

8) Adding 1.5L of acidic antigen repairing solution into a pressure cooker, boiling, placing the tissue slices into the pressure cooker, continuing heating the pressure cooker to exhaust, stopping heating after exhausting for 2 minutes, cooling for 25 minutes by using tap water flow, and washing with PBS for three times.

9) 3% hydrogen peroxide solution (original concentration 30%) is prepared and dropped on the tissue, and after incubation for 20 minutes at normal temperature in the dark, PBS is washed for three times.

10) The tissue was incubated with the same serum working solution as the secondary antibody species dropwise for 40 minutes at room temperature, followed by addition of the primary antibody dilution overnight at 4 ℃.

11) Rewarming for 10 minutes at normal temperature, washing with PBS 3 times, dripping the secondary antibody diluent on the tissue, and incubating for 1 hour at normal temperature.

12) PBS was washed three times, DAB was developed, the development was observed under a microscope to an appropriate degree, and the development was stopped with tap water. Hematoxylin counterstain for 5 minutes, after 10 seconds of hydrochloric acid alcohol differentiation, the tap water was rewetted for 15 minutes.

13) The mixture is dehydrated according to a gradient of 75% ethanol, 95% ethanol, 100% ethanol I and 100% ethanol II, and xylene I and xylene II are transparent. A neutral gum mounting was used. After the specimen was dried in a fume hood, the specimen was read and photographed by a microscope.

Secondly, the result is:

fig. 9 and 10 show that FAM19a5 is highly expressed in mouse brain tissue hippocampus, and exhibits moderate levels of expression in amygdala, thalamus, cerebral cortex, hypothalamus, and cerebellum. FIG. 11 shows immunofluorescent staining of mouse brain tissue, which also demonstrates high expression of FAM19A in the hippocampal region of mouse brain tissue, particularly with relatively strong positive staining on pyramidal neurons.

Example 5 expression of FAM19A5 on different neural cells

The method comprises the following steps:

mice were managed in the same manner as described above. Paraffin sections of mouse brain tissue were obtained as described above. Immunofluorescent staining was then used to detect co-expression of different neural cell specific markers with FAM19a 5. Real-time PCR was used to detect the expression of FAM19A5 in different neural cells. And (3) detecting the expression condition of FAM19A5 in different nerve cell culture supernatants by using double-antibody sandwich microspheres.

And (3) immunofluorescence staining:

1) the paraffin sections are placed in an oven at 60 ℃ for baking for 2 hours to melt wax, then are rapidly placed in xylene I and xylene II to be respectively soaked for 20 minutes, then the paraffin sections are sequentially placed in 100% ethanol I, 100% ethanol II, 95% ethanol and 75% ethanol to be respectively soaked for 5 minutes, and then are placed in PBS.

2) Adding 1.5L of acidic antigen repairing solution into a pressure cooker, boiling, placing the tissue slices into the pressure cooker, continuing heating the pressure cooker to exhaust, stopping heating after exhausting for 2 minutes, cooling for 25 minutes by using tap water flow, and washing with PBS for three times.

3) 0.3 percent TritonX-100 solution is prepared and dripped on the tissue, after the incubation for 20 minutes at normal temperature, PBS is used for washing three times.

4) The tissue was incubated with the same serum working solution as the secondary antibody species dropwise for 40 minutes at room temperature, followed by addition of the primary antibody dilution overnight at 4 ℃.

5) Rewarming for 10 minutes at normal temperature, washing for 3 times by PBS, dripping fluorescent secondary antibody diluent on the tissue, and incubating for 1 hour at normal temperature.

6) PBS was washed three times, DAPI counterstained, and mounted with an anti-quencher. And (5) reading the film by a microscope and taking a picture.

Secondly, the result is:

as shown in fig. 12, FAM19a5 was co-expressed with the neuron specific marker NeuN. As shown in fig. 13, FAM19a5 was co-expressed with neural stem cell specific marker Nestin. As shown in fig. 14, FAM19a5 was co-expressed with the oligodendrocyte-specific marker Olig 2. As shown in fig. 15, FAM19a5 was co-expressed with the glutamatergic neuron specific marker VGLUT 2. The results showed that neurons (particularly glutamatergic neurons), neural stem cells and oligodendrocytes express FAM19a 5. Real-time PCR and double-antibody sandwich microspheres were used to detect the expression of FAM19A5 in 293T, neurons, astrocytes and neural stem cells. The results are shown in fig. 16 and 17, and neurons and neural stem cells express and secrete FAM19a 5.

Example 6 deletion of FAM19A5 results in depression-like behavior in mice

To evaluate the depressive-like behavior of Fam19a5 knockout mice, Fam19a5 was evaluated^-/-Open field experiments, sucrose preference experiments, forced swim experiments, and tail suspension experiments were performed on mice and littermate Wild Type (WT) mice.

Open Field experiment (Open Field Test): the mice were placed individually in the center of a clear box made of plastic 40cm long, 40cm wide and 30cm high. Explore freely for 30 minutes. Meanwhile, the mouse moving track was recorded with Smart 3.0 video system. The total distance, speed of movement and time in the central zone of the first 5 minutes were counted for each mouse moving over the open field.

Sucrose Preference Test (Sucrose Preference Test): mice were housed in a single cage, on which two water bottles were placed, one containing sucrose water at a concentration of 1% (mass/volume) and the other containing sterilized tap water. After 24 hours of acclimation to the two-bottle feeding, the mice began the official experiment and recorded the initial weight of the two water bottles. To avoid the mice having positional preference, the water bottle position was changed every 8 hours. The final weight of the two water bottles was recorded after 24 hours and the amount of water and sucrose taken in by the mice over 24 hours was calculated. The sucrose preference index is calculated as: sucrose preference percentage (%). sucrose intake water/(water intake + sucrose water) × 100%

Forced Swimming Test (Forced Swimming Test): mice were individually placed in a clear acrylic cylinder (20 cm diameter, 40cm height) containing 30cm of water, the temperature of which was maintained at 22 ℃. The state of the mice 6.5 minutes after being placed in water was recorded using the Smart 3.0 video system. Mice were analyzed for time to rest in the last 5 minutes of the test.

Tail overhang Test (Tail Suspension Test): the mouse tail was suspended with tape for 6.5 minutes and the state of the mouse 6.5 minutes after suspending the tail was recorded using Smart 3.0 video system. Mice were analyzed for time to rest in the last 5 minutes of the test.

Secondly, the result is:

open field experiments are used to measure the distance and time of movement of an animal in the central or peripheral area of an open space, the distance of movement reflecting the amount of spontaneous activity of the animal in the open field, and the time of movement in the central area reflecting the extent of anxiety or depression of the animal. The results show that Fam19a5 was measured within 30 minutes of the open field test^-/-The total distance and speed of movement of the mice were significantly increased compared to WT mice (fig. 18 and 20), whereas within the first 5 minutes of the experimental measurement, Fam19a5^-/-The percentage of time that the mice occupied the central zone was significantly reduced compared to WT mice (figure 19). This experiment shows Fam19a5 compared to littermate WT mice^-/-Mice show high activity and significantly increased anxiety/depressive-like behavior. To go further forwardFam19a5^-/-Whether mice exhibit depression-like behavior, we performed sucrose preference experiments, forced swim experiments, and tail suspension experiments. We first performed a Sucrose Preference Test (SPT) that measures the percentage of animals' intake of a 1% Sucrose (w/v) solution, reflecting whether the animal has anhedonia, lack of interest in reward stimuli, and like depressive-like manifestations. The results show that Fam19a5^-/-The preference of the mice for sucrose solution was significantly lower than that of the WT control mice (fig. 21), indicating Fam19a5^-/-The anhedonia behavior of the mice was significantly increased. We then performed a Forced Swimming Test (FST) that measures the immobility time of the animal in water as an acute stress test reflecting the degree of depression of the animal. The results show that Fam19a5^-/-Mice showed longer immobility time (FIG. 22), reflecting Fam19a5^-/-The mice had longer despair behavior. This result was also confirmed in the Tail Suspension Test (TST), another acute stress test, Fam19a5^-/-Mice also exhibited longer immobility time (fig. 23). The above results show that Fam19a5^-/-The mice showed increased depression-like behavior compared to littermate WT mice.

Example 7 deletion of FAM19A5 results in cognitive impairment in mice

To evaluate the cognitive behavior of Fam19a5 knockout mice, Fam19a5 was tested^-/-The water maze experiment was performed in mice and littermate Wild Type (Wild Type, WT) mice.

Water Maze experiment (Morris Water Maze, MWM): the water maze used in this study consisted of a circular water box with a diameter of 120cm and a depth of 60 cm. The water tank is filled with opaque water, the temperature is 22 +/-2 ℃, and a transparent escape platform with the diameter of 10cm is arranged and is positioned 1cm below the water surface. And pasting different visual pictures around the water tank to be used as space marks. A camera is arranged above the water maze and records the swimming track of the mouse in the water maze. The water maze is divided into four quadrants, a target quadrant (the quadrant previously containing the platform) and three non-target quadrants (the opposite quadrant, the adjacent right quadrant and the adjacent left quadrant). On day 0, the mouse was trained to find a platform with a visible flag. On days 1-6, the platform was submerged 1-2cm below the water surface, the mouse was placed in one of the four quadrants of the maze facing the wall of the water maze, and the platform was searched for 60 seconds. If one mouse fails, it is directed to the platform and maintained for 15 seconds. Four experiments were performed daily, with each experiment being separated by 45 minutes. Escape latency was recorded for each mouse for each experiment. On day 7, the platform was removed, the spatial memory test was performed, the time (%) of the mouse in the target quadrant was compared to all other quadrants, and the number of times the mouse crossed the platform in the target quadrant was compared to similar areas in all other quadrants.

Secondly, the result is:

in the MWM experiment, on day 0, the mouse can see the visible flag on the platform, Fam19a5^-/-There was no significant difference in the time for the mice to find the platform compared to WT mice, indicating Fam19a5^-/-The motivation for mice to find the platform was not different compared to WT mice (fig. 24). Day 1-6, Fam19a5^-/-Mice found a significant increase in latency of the hidden platform during training to find the underwater platform (figure 24). Day 7, after platform withdrawal, Fam19a5^-/-Mice occupied significantly less time in the target quadrant where the platform was located compared to WT mice (fig. 25 and 26). Furthermore, Fam19a5^-/-Mice crossed the platform region significantly less frequently than WT mice (FIG. 27), further suggesting that Fam19a5^-/-Mice exhibit cognitive impairment of spatial learning and memory.

Example 8 Chronic stress-induced Depression causes significant reductions in mouse plasma and Hippocampus FAM19A5 expression

The chronic stress model in mice is thought to better reflect depression in humans. To explore whether the expression of FAM19a5 changes in depression, wild-type mice were subjected to three animal behavioral-verified stress models, namely Chronic Restraint Stress (CRS), Chronic Forced Swimming Stress (CFSS) and Chronic social frustration stress (CSDS), to explore the expression change of FAM19a 5.

Chronic Restraint Stress (CRS): male C57BL/6J mice, 2-3 months of age, were placed in a 50mL centrifuge tube with three vents, allowing them to stretch the limbs, but not allow them to move within the tube. Chronic restraint stress was applied for 2-3 hours per day for 21 consecutive days. Age-matched non-stressed animals served as controls.

Chronic Forced Swimming Stress (CFSS): male C57BL/6J mice, 2-3 months of age, were placed in clear acrylic cylinders 40cm high for 5 consecutive days with 5 minutes of chronic forced swimming stress applied daily. Age-matched non-stressed animals served as controls.

Chronic Social Defeat Stress (CSDS): the male C57BL/6J mice 6-8 weeks old are directly placed in cages of male CD-1 retired mice 4-6 months old, and social contusion stress is carried out for 5-10 minutes every day for 10 days. After direct contact with CD-1 challenged mice, C57BL/6J mice were transferred to the other side of the cage, separated from each other by a perforated partition plate to prevent physical contact, and placed in separate cages for 24 hours. Control groups C57BL/6J pairs of mice were placed in the same cages. Control mice were rotated daily in control cages. Social interaction testing was performed 24 hours after the last social frustration stress. C57BL/6J mice were habituated to the social interaction test apparatus 1 hour prior to the social interaction test. In the first 2.5 minutes of the experiment ("no target"), the C57BL/6J socially frustrated mice were placed in a social interaction device, allowed to explore freely an open field (40 × 40cm) with an empty wire cage (10 × 6cm) without "target" CD-1 mice. In a second 2.5 minute trial ("targetted"), with the "target" CD-1 mouse inside the wire cage, the mouse was replaced into the device, allowing it to move freely. The times at the "corner area" (9 x 9cm) and the "interaction area" (12 x 25cm) were recorded and analyzed using a Smart 3.0 video tracking system. The social interaction ratio refers to the ratio of time spent in the "interaction area" to the time spent in the "corner area". The recovery type means that the social interaction ratio is more than or equal to 1, and the susceptible type means that the social interaction ratio is less than 1.

Secondly, the result is:

first, we induced CRS and CFSS models, and after the last confinement or forced swimming, we harvested plasma from model and control mice and isolated hippocampus of the mice. And the expression level of FAM19A5 was detected using Real-time PCR and a microsphere system. The results showed that the mRNA and protein levels of FAM19a5 were significantly down-regulated in plasma and hippocampus of model mice induced by chronic restraint stress or chronic forced swimming stress (fig. 28, 29 and 30). We then induced the CSDS model and tested the levels of FAM19a5 in plasma and hippocampus of model mice after social interaction testing. FAM19a5 levels were significantly reduced in plasma in susceptible mice (fig. 31) and also FAM19a5 protein levels in hippocampus (fig. 32) compared to control and recovery mice, consistent with CRS and CFSS mice results. The above results indicate that FAM19a5 levels in plasma and hippocampus were significantly reduced under chronic stress conditions.

Example 9 AAV9-FAM19A5 reduces chronic stress-induced depressive-like behavior in mice with antidepressant drug activity

To further explore the role of FAM19A5 in depression, Shandong Weizhen bioscience, Inc. was entrusted to construct human FAM19A5(hFAM19A5) polynucleotide sequence (SEQ ID No: 1) into adeno-associated virus AAV9 containing neuron-specific promoter hSyn to obtain AAV9-hSyn-hFAM19A5, and AAV9-hSyn-hFAM19A5 (shown in figure and description as AAV-FAM19A5) was injected in situ into hippocampus to over-express human FAM19A5 protein (SEQ ID No: 2) in chronic restraint stress-induced depression model mice to study the effect of human FAM19A5 protein on the depression behavior of mice.

In situ injection of virus into hippocampus:

the mice were anesthetized by intraperitoneal injection of sodium pentobarbital (50mg/kg) and fixed in a stereotaxic apparatus. When fixed, press the horizontal pole gently with mouse anterior teeth card on mouse adapter anterior teeth clamp earlier, around the adjustment mouse with horizontal position, hold up the mouse head, will control the side ear pole and insert mouse external auditory canal, adjust the ear pole and make the mouse head keep at central point and put, screw the ear pole screw for the mouse head can not rock, screws up the anterior teeth simultaneously and presss from both sides the screw. The hair on the mouse head was removed with a razor, the exposed skin on the head was disinfected with 75% ethanol, the skin was cut open, and the connective tissue on the surface of the skull was removed by wiping with a dry cotton swab, exposing the front and back fontanels.

Mineral oil is injected into the glass electrode and is installed on the injection pump, and the injection pump is connected to the stereotaxic apparatus. The front and back fontanels positions were determined and the mouse head was leveled. The location of the nucleus of interest was determined by moving the syringe pump according to the coordinates of the CA1 region of the hippocampus (Bregma Antifoperosior (AP): 2.06mm, Media (ML): 1.50mm,. + -. 1.50mm, Dorsoventral (DV): 1.30mm), and Bregma 0. The skull is carefully thinned slowly with a burr drill at the injection site and the milling is stopped when a small window appears in the skull. The periphery of the skull window was gently wiped with a medical cotton swab to remove impurities. The 500nL titer is 1X 10 by suction with a syringe pump¹³The AAV9 viral vector (AAV-FAM19A5) containing the neuron-specific promoter or an equivalent control unloaded virus (AAV-Null) having the same titer, and then the position of the glass electrode was finely adjusted to be the same as that of the objective nucleus, and the glass electrode was slowly lowered to the objective depth. The micro-syringe pump is set to complete the injection of the virus within 5 minutes and stay for 5 minutes to fully diffuse the virus and prevent the virus from leaking out of the nuclear mass along with the extraction of the glass electrode.

After 3 weeks of virus expression, the two groups of mice were induced for 10 days in the same manner as above for 3 hours per day. After the model is finished, the open field experiment and the tail suspension experiment of the same method are carried out after the mice recover for 1-2 days.

Secondly, the result is:

in the open field experiment, the percentage of time in the central zone and the number of entries into the central zone of mice infected with AAV-FAM19a5 increased significantly compared to AAV-Null control mice within the first 5 minutes of the experimental measurement (fig. 33 and 34), indicating that AAV-FAM19a5 mice had reduced depressive-like behavior. The results of tail suspension experiments showed that mice infected with AAV-FAM19A5 showed shorter immobility time (FIG. 35), reflecting the shorter desperate behavior of mice infected with AAV-FAM19A 5. Because the FAM19A5 gene on the AAV vector expresses the Flag label at the same time, the expression level of Flag in the CA1 area of mouse hippocampus infected by AAV-Null and AAV-FAM19A5 is detected by immunofluorescence to reflect the expression condition of FAM19A5, and the expression level of FAM19A5 in mouse hippocampus is detected by a microsphere system. As a result, the protein level of FAM19A5 in the hippocampus of mice infected with AAV-FAM19A5 was found to be significantly higher than that of the control mice (FIG. 36 and FIG. 37), indicating successful overexpression. The results show that compared with AAV-Null control mice, AAV-FAM19A5 over-expressing FAM19A5 can reduce depression-like behaviors of the mice, and AAV-FAM19A5 has antidepressant activity.

Example 10 deletion of FAM19A5 affects the composition of immune cells in mouse peripheral blood and brain

To assess the different immune cell ratios in peripheral blood and brain of Fam19a5 knockout mice, Fam19a5 was assessed using flow cytometry^-/-Peripheral blood and brain of mice and littermate Wild Type (Wild Type, WT) mice were evaluated for different immune cell ratios.

Detection of immune cells in the brain:

the whole brain tissue of the mouse is put into HBSS solution to be ground and is paved into 7mL PBS solution containing 2% FBS to prepare single cell suspension of the brain. 7mL of the slurry was mixed with 3mL of 90% Percoll solution and slowly added dropwise to a 15mL centrifuge tube to which 70% Percoll had been previously added. Centrifuge at 18 ℃ for 30 minutes at 500g without brake. Cells were washed twice with 3 volumes of HBSS solution. PBS containing 2% FBS was used as a blocking solution and blocked on ice for 20 minutes. According to the instruction, adding a proper amount of staining solution containing a fluorescence labeling flow antibody, placing on ice, and incubating for 30 minutes in a dark place. The mixture was centrifuged at 2000rpm for 5 minutes at 4 ℃ and the supernatant was discarded. Washed 1 times with PBS, resuspended in 300. mu.L PBS and examined on a BD Verse flow cytometer. The data obtained were processed and analyzed with the software FlowJo 7.6.

Detection of immune cells in peripheral blood:

100. mu.L of peripheral blood of a mouse was added to 1mL of PBS solution and mixed well. Centrifuge at 4500rpm for 5 min at 4 ℃ and discard the supernatant. Add 700. mu.L of ACK red blood cell lysate to lyse red for 3 minutes on ice. After neutralization by adding 700. mu.L of PBS solution, the mixture was centrifuged at 4500rpm for 5 minutes at 4 ℃ and the supernatant was discarded. After repeating the red splitting once, washing with PBS 1 time. PBS containing 2% FBS was used as a blocking solution and blocked on ice for 20 minutes. According to the instruction, adding a proper amount of staining solution containing a fluorescence labeling flow antibody, placing on ice, and incubating for 30 minutes in a dark place. The mixture was centrifuged at 2000rpm for 5 minutes at 4 ℃ and the supernatant was discarded. Washed 1 times with PBS, resuspended in 300. mu.L PBS and examined on a BD Verse flow cytometer. The data obtained were processed and analyzed with the software FlowJo 7.6.

Secondly, the result is:

fam19a5 compared to WT mice^-/-CD4 in peripheral blood of mice⁺T lymphocyte proportion was significantly up-regulated and B lymphocyte proportion was significantly down-regulated (FIG. 38), whereas Fam19a5^-/-The monocyte/macrophage ratio in the mouse brain was significantly up-regulated (figure 39).

Example 11 FAM19A5 protein can cross the blood brain barrier

The method comprises the following steps:

to investigate whether FAM19A5 eukaryotic proteins could pass the blood brain barrier, we administered Fam19a5^-/-Mice were injected intravenously (i.v.) or intraperitoneally (i.p.) with 5 μ g of FAM19A5 eukaryotic protein, and after 5 minutes, mice were injected intraperitoneally with sodium pentobarbital (50mg/kg) for anesthesia, and mouse plasma and cerebrospinal fluid were collected, and the content of FAM19A5 was measured using a microsphere detection system.

Secondly, the result is:

FAM19a5 protein was detected in the plasma of mice 5 minutes after either tail vein injection or intraperitoneal injection of protein, whereas FAM19a5 protein was detected in the cerebrospinal fluid of mice only 5 minutes after tail vein injection (fig. 40). These results indicate that FAM19a5 eukaryotic proteins can pass the blood brain barrier, suggesting that peripheral FAM19a5 may play a role in the brain.

Example 12 Fam19a5 knock-out mice with increased microglial Iba-1 expression in brain

The method comprises the following steps:

mice were managed in the same manner as described above, and then brain tissue frozen sections were obtained. WT and Fam19a5 were then detected by immunofluorescence staining^-/-Neuron specific marker NeuN and microglia specificity in mouse cortex and hippocampusStaining of the sexual marker Iba-1.

Immunofluorescence:

1) material taking: after anesthetizing the mice with sodium pentobarbital, the hearts were perfused with cold PBS and 4% Paraformaldehyde (PFA). Mouse tissues are taken, tissue blocks are trimmed to a proper size and then are placed into a paraformaldehyde solution for fixation at 4 ℃ for 24 hours.

2) And (3) dehydrating: the tissue was taken out from the polyoxymethylene solution, washed with PBS, and then dehydrated in 20% and 30% sucrose solutions for 24 hours at 4 ℃.

3) Embedding: the tissue is put into a module made of tin foil paper, and after an OCT embedding medium is added, the tissue is put into a refrigerator at minus 80 ℃ for solidification and molding.

4) Slicing: the prepared tissue blocks were sectioned using a Leica 3050s semi-automatic microtome, 50 μm thick, using slides pre-treated with cations. The attached frozen sections were dried in an oven at 37 ℃ overnight and then ready for use.

5) The stored frozen sections were washed 3 times in PBS to dissolve the OCT gel, 5 minutes each time.

6) The incubation was performed for 20 minutes by adding PBS containing 0.3% Triton X-100.

7) After washing with PBS 3 times, the goat serum working solution was added and sealed at room temperature for 1 hour.

8) Primary antibody was added and incubated overnight at 4 ℃.

9) After rewarming for 10 min at room temperature, PBS was washed 3 times, Alexa 488 or Alexa 594-coupled secondary IgG was added and incubated for 1 h at room temperature in the dark.

10) Washed 3 times with PBS, counterstained with nuclear dye DAPI, and incubated for 20 minutes at normal temperature in the dark.

11) Washing with PBS for 3 times, sealing with anti-fluorescence quenching sealing agent, observing under laser confocal microscope, and taking pictures.

Second, result in

Fam19a5^-/-The positive staining of Iba-1 was more pronounced and morphologically altered in the mouse cortex and hippocampus (FIG. 41). And (4) prompting: FAM19a5 may affect the morphology and number of microglia.

Example 13 inhibition of the inflammatory phenotype of the microglia line BV2 by FAM19A5

The method comprises the following steps:

to explore the effect of FAM19a5 on microglial function, we used Real-time PCR to examine the inflammatory phenotype of microglial line BV2 after addition of FAM19a5 eukaryotic protein and LPS.

Microglia cell line polarization experiments:

1) cells were plated in 6-well plates overnight at approximately 6X 10 per well⁵(ii) individual cells;

2) PBS washing twice, adding 1 ug/mL LPS, and adding 200ng/mLFAM19A5 eukaryotic protein;

3) after 6 hours, washing twice with PBS, collecting cells, adding Trizol, and extracting RNA;

4) Real-timePCR detects the mRNA expression levels of IL-1 β, IL-6, TNF- α and iNOS.

Second, result in

FAM19A5 inhibited the mRNA expression levels of IL-1 β, IL-6, TNF- α in microglia line BV2 after LPS stimulation (FIG. 42). The above results show that: FAM19a5 inhibited the inflammatory phenotype of the microglia line BV 2.

Example 14 inhibition of proliferation and self-renewal of mouse neural stem cells by FAM19A5

The method comprises the following steps:

to explore the effect of FAM19a5 on neural stem cell function, we removed embryonic day 16.5 wild-type mouse brains, isolated neural stem cells in vitro for culture, and then administered different concentrations of FAM19a5 recombinant protein stimulation to the culture supernatant, and observed the effect of FAM19a5 on neural stem cell proliferation and self-renewal after 5 days.

Neural stem cell proliferation and self-renewal experiments:

neural stem cells (neurospheres) cultured for 3-5 days were collected, digested with Accutase, and then seeded at 3000 cells/well in 96 plates overnight. The cells were washed twice with PBS, the cells were exchanged, and different concentrations of FAM19A5 recombinant protein were added and cultured for 24 hours. After CCK-8 was added, the mixture was incubated in an incubator for 2 to 4 hours, and the absorbance OD450 was measured.

Secondly, the result is:

FAM19A5 can inhibit proliferation of mouse primary nerve stem cell (neurosphere), and inhibit secondary nerveThe number and size of the spheres, and the inhibitory effect, was dependent on the concentration of FAM19a5 (fig. 43). To further validate the inhibitory effect of FAM19a5 on neural stem cell proliferation and self-renewal, we removed 16.5 day embryos of WT and FAM19a5^-/-Mouse brain, in vitro neural stem cell isolation for culture, subsequent stimulation with different concentrations of FAM19A5 recombinant protein in culture supernatant, 5 days later observation of WT and FAM19a5^-/-Proliferation and self-renewal of mouse neural stem cells. The results show Fam19a5^-/-The proliferation capacity of the mouse primary neural stem cell is enhanced, the number and the size of the second generation neurospheres are increased, and the FAM19A5 recombinant protein can reverse the proliferation and self-renewal capacity of the knockout mouse primary neural stem cell (figure 44). The above results show that: FAM19A5 can inhibit proliferation and self-renewal of mouse neural stem cells.

SEQUENCE LISTING

<110> Beijing university

<120> medical use of FAM19a5

<130> WX2021-BY-002

<150> CN 202010259573.1

<151> 2020-04-03

<160> 8

<170> PatentIn version 3.5

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