Application of myeloid cell trigger receptor 2 in preparation of sepsis related functional products

文档序号：1874543 发布日期：2021-11-23 浏览：6次中文

阅读说明：本技术 髓系细胞触发受体2在制备脓毒症相关功能产品中的应用 (Application of myeloid cell trigger receptor 2 in preparation of sepsis related functional products ) 是由黄曦明思奇吴永坚王之影于 2021-05-19 设计创作，主要内容包括：本发明涉及生物医药技术领域,公开了髓系细胞触发受体2在制备脓毒症相关功能产品中的应用。本发明揭示了TREM-2在脓毒症诊断和治疗中的作用。依据包括两方面,一方面TREM-2在脓毒症患者中表达升高且与患者病情密切相关。另一方面,阻断TREM-2可缓解脓毒症小鼠的症状,并提高小鼠的生存率。该发明为临床脓毒症诊断、干预和治疗提供了良好的策略,具有十分广阔的应用前景。(The invention relates to the technical field of biological medicines, and discloses application of a myeloid cell trigger receptor 2 in preparation of functional products related to sepsis. The invention discloses the role of TREM-2 in sepsis diagnosis and treatment. According to two aspects, on the one hand, TREM-2 is expressed in patients with sepsis and is closely related to the condition of the patients. On the other hand, blocking TREM-2 can alleviate symptoms of sepsis mice and increase survival rates of mice. The invention provides a good strategy for clinical sepsis diagnosis, intervention and treatment, and has very wide application prospect.)

Use of TREM-2 in the preparation of a formulation for the diagnosis of sepsis, wherein the formulation is used to detect the expression of TREM-2 and the expression of TREM-2 is positively correlated with sepsis.

Use of TREM-2 in the preparation of a formulation for the prognostic assessment of sepsis, wherein the formulation is used to detect the expression of TREM-2 and the expression of TREM-2 is positively correlated with the severity of the disease in patients with sepsis.

3. The use of claim 1 or 2, wherein the agent comprises an antibody capable of specifically binding to TREM-2, or a ligand for TREM-2.

4. Use according to claim 1 or 2, wherein the TREM-2 is from CD11b⁺And/or CD3⁺T cells.

Use of TREM-2 in the manufacture of a functional product for the treatment/amelioration of sepsis, wherein said functional product has the function of blocking TREM-2.

6. The use according to claim 5, wherein the functional product comprises: one or more of a TREM-2 protein inhibitor, a TREM-2 gene deficient or silenced immune-related cell, a differentiated cell thereof, or a gene recombination construct.

7. The use according to claim 6, wherein the functional product comprises:

(i) an activated antibody, nucleotide, lentivirus or adenovirus which takes a TREM-2 transcript as a target sequence and can activate the expression of a TREM-2 gene expression product or gene transcription;

(ii) a construct containing a TREM-2 complementary sequence and capable of forming an activator molecule which promotes expression of a TREM-2 gene expression product or gene transcription upon transfer into the body;

(iii) immune-related cells, differentiated cells thereof, or constructs following activation of a TREM-2 gene sequence.

8. The use of claim 7, wherein the functional product is selected from the group consisting of a TREM-2 small molecule inhibitor, a TREM-2-Fc fusion protein, a TREM-2 specific blocking antibody, a TREM-2 targeting siRNA, a TREM-2 knock-out or silencing agent and immune cell, and a TREM-2 gene-deficient mouse derived immune cell.

9. Use according to any one of claims 5 to 8, wherein the treatment/alleviation comprises at least one of the following:

(1) increasing sepsis survival rate;

(2) delay of progression of sepsis disease;

(3) reducing inflammatory factor production caused by sepsis;

(4) relieving organ damage caused by sepsis;

(5) relieving the fatty acid oxidative metabolism disorder caused by sepsis.

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of a myeloid cell trigger receptor 2 in preparation of functional products related to sepsis.

Background

Sepsis is a clinical syndrome of dysfunction of organs that endanger life due to a disturbance of the immune response of the body caused by infection, and the main causes include infection, trauma, surgery, burns, and the like. Sepsis can lead to severe complications such as septic shock and multiple organ failure in the late stage of development, making it one of the critical diseases with high clinical mortality. Recent data show that 3150 million people worldwide per year develop sepsis, with up to 530 million deaths. Despite the increasing survival of patients with sepsis in recent years, the long-term mortality rate of sepsis still reaches 20% -50%, and if septic shock develops, the mortality rate increases to 40% -50%. At the same time, health care expenditures for sepsis are rising, with annual costs for sepsis therapy reaching 170 billion in the united states alone. Sepsis has therefore become a significant public health problem worldwide. In the face of such serious disease threats, it is urgently needed to deeply study the pathogenesis of sepsis and find effective therapeutic control measures.

A myeloid cell Triggering Receptor (TREM) is a transmembrane receptor expressed on the cell surface of a myeloid cell, belongs to an immunoglobulin superfamily, and consists of an extracellular single immunoglobulin variable region, a positively charged transmembrane region and a short intracellular segment. TREM receptors are localized to 6P21 and 17C3 on human and murine chromosomes, respectively, and are expressed predominantly in myeloid cells, especially monocytes and neutrophils, as well as Natural killer cells (NKs), granulocytes, and dendritic cells, but are expressed less on lymphocytes. Among them, TREM-2 is of great interest because it plays an important role in controlling neurodegenerative diseases, tumors, autoimmune diseases, and other diseases. TREM-2 is involved in regulating various cellular functions, including cell proliferation and survival, phagocytosis, neural development, and inflammatory response. TREM-2 missense mutation can promote the occurrence of nervous system degenerative diseases. Microglia and macrophages deficient in TREM2 have reduced phagocytic capacity for neurons, cell debris and bacteria. In addition, TREM-2 recognizes and binds to apolipoproteins, playing an important regulatory role in lipid metabolism. The exact ligand of TREM2 is not known at present, but substances reported to bind to TREM-2 include dextran, lipopolysaccharide, heat shock protein, apolipoprotein, etc. These studies all show that TREM-2 has great promise as an immunotherapeutic target.

At present, clinical sepsis treatment mainly aims at symptomatic treatment, and no specific treatment medicine exists. Sepsis involves very complex body reactions involving many aspects including physiological processes such as immune response and cellular metabolism. Elucidating the pathogenic mechanism of sepsis and finding an interventable target is crucial for sepsis therapy. In order to improve the survival rate of patients with sepsis, a new immune checkpoint molecule needs to be searched to improve the diagnosis efficiency and the prognosis evaluation accuracy of sepsis and increase the treatment effect of sepsis.

Disclosure of Invention

In order to overcome the above-mentioned drawbacks posed by the prior art, the primary object of the present invention is to provide the use of TREM-2 in the preparation of a formulation for the diagnosis of sepsis.

The second purpose of the invention is to provide the application of TREM-2 in preparing a preparation for sepsis prognosis evaluation.

The third purpose of the invention is to provide the application of TREM-2 in preparing a preparation for treating sepsis.

The purpose of the invention is realized by the following technical scheme:

use of TREM-2 in the preparation of a formulation for diagnosing sepsis, said formulation being for detecting the expression of TREM-2, and the expression of TREM-2 being positively correlated with sepsis.

Use of TREM-2 in the preparation of a prognostic assessment formulation for sepsis, wherein the formulation is used to detect the expression of TREM-2 and the expression of TREM-2 is positively correlated with the severity of the disease in a sepsis patient.

On the one hand, the research of the invention finds that: 1. the expression of the peripheral blood mononuclear cells and T lymphocyte TREM-2 of the sepsis patient is increased, and the TREM-2 can be used as an auxiliary diagnosis index of the sepsis; 2. the expression of TREM-2 is closely related to the disease of a patient suffering from sepsis, and the TREM-2 can be used as a prognostic evaluation index of the sepsis.

Preferably, the sepsis includes bacterial sepsis, viral sepsis, fungal sepsis, sepsis due to trauma, septic shock, and various complications due to sepsis; the diagnostic reagent has the function of diagnosing whether the patient suffers from sepsis or evaluating the severity of the patient's disease based on the expression of TREM-2; the therapeutic agent can achieve the effects of relieving sepsis symptoms and improving survival rate by blocking TREM-2.

Thus, in practice, a subject can be differentially diagnosed as suffering from sepsis by detecting the expression of TREM-2. Meanwhile, the severity of the disease of the patient can be judged according to the expression level of TREM-2, so that the prognosis of the sepsis patient can be evaluated.

Preferably, the formulation comprises an antibody capable of specifically binding to TREM-2, or a ligand for TREM-2.

More preferably, the TREM-2 is from CD11b⁺And/or CD3⁺T cells.

In another aspect of the invention, the research finds that: 1. in a sepsis mouse model, the TRME-2 knockout can effectively improve the survival rate of mice; 2. the thickening of pulmonary alveolar walls of the TREM-2 knockout sepsis mice is reduced, the structural integrity of pulmonary alveoli is increased, and inflammatory infiltration is relieved; 3. the knockout of TRME-2 can inhibit the generation of inflammatory factors in serum and organ grinding fluid of a sepsis mouse; 4. the accumulation of serum and liver triglyceride of a TREM-2 knockout sepsis mouse is reduced, the expression of molecules related to fatty acid oxidation is increased, and the oxidation rate of macrophage fatty acid is increased.

Therefore, the purposes of delaying disease progression, improving survival rate, reducing inflammatory factor production, improving fatty acid oxidative metabolism and reducing organ damage caused by sepsis can be achieved by blocking TREM-2.

Therefore, the invention also provides the application of TREM-2 in preparing a functional product for treating/relieving sepsis, wherein the functional product has the function of blocking TREM-2.

Preferably, the functional product comprises: one or more of a TREM-2 protein inhibitor, a TREM-2 gene deficient or silenced immune-related cell, a differentiated cell thereof, or a gene recombination construct.

More preferably, the functional product comprises:

(iii) immune-related cells, differentiated cells thereof, or constructs following activation of a TREM-2 gene sequence.

Specifically, the functional product is selected from a TREM-2 small molecule inhibitor, a TREM-2-Fc fusion protein, a TREM-2 specific blocking antibody, a TREM-2 targeting siRNA, a TREM-2 knockout or silencing reagent, an immune cell and a TREM-2 gene-deficient mouse derived immune cell.

Preferably, the treatment/alleviation includes at least one of the following:

(1) increasing sepsis survival rate;

(2) delay of progression of sepsis disease;

(3) reducing inflammatory factor production caused by sepsis;

(4) relieving organ damage caused by sepsis;

(5) relieving the fatty acid oxidative metabolism disorder caused by sepsis.

Compared with the prior art, the invention has the following beneficial effects:

the invention discloses the role of TREM-2 in sepsis diagnosis and treatment. According to two aspects, on the one hand, TREM-2 is expressed in patients with sepsis and is closely related to the condition of the patients. On the other hand, blocking TREM-2 can alleviate symptoms of sepsis mice and increase survival rates of mice. The invention provides a good strategy for clinical sepsis diagnosis, intervention and treatment, and has very wide application prospect.

Drawings

FIG. 1 correlation analysis shows that TREM-2 is up-regulated in peripheral blood mononuclear cells and T lymphocytes from patients with sepsis;

FIG. 2 correlation analysis shows that the expression of TREM-2 in peripheral blood mononuclear cells from patients with sepsis is closely related to the patient's condition;

FIG. 3 shows that, in a sepsis mouse model, knockout of TRME-2 is effective in increasing mouse survival;

FIG. 4 shows that TREM-2 knockout sepsis mice have reduced alveolar wall thickening in the lungs, increased alveolar structural integrity, and reduced inflammatory infiltration;

FIG. 5 shows that the knockout of TRME-2 inhibits the production of inflammatory factors in serum and organ slurry of septic mice;

FIG. 6 shows that serum and liver triglyceride accumulation is reduced, fatty acid oxidation-related molecule expression is increased, and macrophage fatty acid oxidation rate is increased in TREM-2 knockout sepsis mice.

Detailed Description

The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The test methods used in the following experimental examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Example 1 expression of TREM-2 in peripheral blood mononuclear cells and T lymphocytes from patients with sepsis

Collecting peripheral blood of 46 healthy volunteers and 54 patients with sepsis, separating PBMC by density gradient centrifugation, and detecting TREM-2 in CD11b by flow method⁺Monocytes and CD3⁺Expression levels on T lymphocytes.

Collecting peripheral blood of healthy people and sepsis patients 5mL each, lysing erythrocytes to prepare single cell suspension, adjusting total cell amount to 5x10⁶Cells, adding TREM-2 antibody (R) to 100. mu.L system&Company D, cat 1278P) and antibodies to CD11b and CD3 (both from Biolegend), were mixed and protected from light for 30 minutes, and flow-assayed by FCS/SSC gating analysis for the proportion of TREM-2 positive cells to CD11b and CD3 positive cells, respectively.

As shown in figure 1, it is TREM-2 CD11b in peripheral blood of healthy people and patients with sepsis⁺And CD3⁺Expression level profile on the surface of T cells. Let the door be CD11b⁺And CD3⁺Cells, it can be seen that TREM-2 in septic patients is CD11b compared to healthy humans⁺And CD3⁺Expression in cells is significantly upregulated.

The above results illustrate that: the expression of TREM-2 in the peripheral blood of a patient with the sepsis is increased, which indicates that whether the patient suffers from the sepsis can be judged in an auxiliary way by detecting the expression of TREM-2 in the peripheral blood of the patient, namely, TREM-2 can be used as an auxiliary diagnostic index of the sepsis.

EXAMPLE 2 correlation of the expression of TREM-2 in peripheral blood mononuclear cells from patients with sepsis with the patient's condition

Samples were collected on the Day of admission (Day 0) and after 1 Day of treatment (Day 1), 3 days (Day 3), 5 days (Day 5), 7 days (Day 7) of septic patients, and CD11b was flow-assayed⁺Analyzing TREM-2 expression of cells⁺CD11b⁺The proportion of cells. Meanwhile, clinical data of a patient are collected, and the correlation between TREM-2 expression and sepsis condition monitoring indexes and organ function damage indexes is analyzed.

The results in FIG. 2A show that after the patient is hospitalized and the symptoms are controlled, the level of serum acute C-reactive protein (CRP) is continuously reduced (CRP is an important index for clinical sepsis diagnosis, reflects the inflammatory condition of the body and can be used as an index for evaluating the severity of the disease), and CD11b is simultaneously added⁺Cellular TREM-2 expression also shows a downward trend, suggesting that TREM-2 expression may be correlated with patient condition.

The results in FIG. 2B show that the expression of TREM-2 in sepsis patients has a positive correlation with the inflammation index CRP.

The results in FIG. 2C show that TREM-2 expression in sepsis patients is positively correlated with alanine amino transfer (ALT), an organ injury indicator.

FIG. 2D shows that TREM-2 expression in sepsis patients is positively correlated with the kidney injury indicator urea nitrogen (BUN).

The results in FIG. 2E show that TREM-2 expression in patients with sepsis is positively correlated with Total bilirubin (Total bilirubin), an index of liver damage.

These results indicate that CD11b⁺The expression of the TREM-2 cell is closely related to the disease condition of a patient with sepsis, and the higher the expression of the TREM-2 cell is, the more serious the disease condition of the patient is, and the worse the prognosis is. The close correlation between the TREM-2 expression and the disease progression of a patient suffering from sepsis is also demonstrated, which indicates that the TREM-2 can be used as a prognostic evaluation index of the sepsis and is expected to influence the generation and development of the sepsis by interfering the TREM-2 expression.

Example 3 Effect of knockout of TREM-2 on mouse survival

Wild Type (WT) mice and TREM-2 gene knockout (TREM-2) using 4-6 week SPF grade female C57BL/6 background^-/-) And (3) respectively constructing a Cecum Ligation and Perforation (CLP) sepsis model, a Lipopolysaccharide (LPS) induced sepsis mouse model and a Pseudomonas Aeruginosa (PA) infected sepsis model, and observing and recording the survival rate of the mouse so as to determine the influence of TREM-2 on the overall outcome of sepsis.

Figure 3A shows that knockout of TREM-2 can significantly improve survival of septic mice in the CLP model.

FIG. 3B results show that TREM-2 compares to WT mice in a mouse model of LPS sepsis^-/-The survival rate of the mice is obviously improved.

The results in FIG. 3C show that in a sepsis mouse model with bacterial (PA bacterial) infection, knockout of TREM-2 can significantly improve mouse survival.

In conclusion, it can be seen that: the TREM-2 is knocked out from the sepsis mouse, so that the survival rate of the mouse can be effectively improved, and the probability of reducing the death rate of patients suffering from sepsis by blocking the TREM-2 is prompted.

Example 4 Effect of knockout TREM-2 on Lung inflammatory infiltration and injury in septic mice

LPS-induced sepsis models were constructed using 4-6 week SPF grade female C57BL/6 mice. The mice were sacrificed 12 hours after construction, the lung leaflets were fixed with 4% paraformaldehyde, H & E staining was performed after paraffin-embedded sections, and the mice were observed under a microscope for lung inflammatory infiltration and injury, the results of which are shown in fig. 4.

The results indicate that the alveolar structural integrity of WT sepsis mice was destroyed, alveolar walls thickened, and a large number of inflammatory cells were infiltrated. And TREM-2^-/-The mouse has reduced alveolar wall thickening, increased alveolar structural integrity and reduced inflammatory infiltration of the lung.

It follows from this that: TREM-2 knockout can reduce inflammatory infiltration and pathological damage in the lungs of septic mice, leading to reduced alveolar wall thickening, increased alveolar structural integrity, and reduced inflammatory infiltration. The results suggest that organ damage caused by sepsis can be alleviated by blocking TREM-2.

Example 5 Effect of knockout TREM-2 on inflammatory factor production in septic mice

The results in FIG. 5A show that TREM-2 knockout significantly reduces the level of the inflammatory factor IL-1 β in plasma and liver and lung lapping fluid.

FIG. 5B shows the results, TREM-2^-/-The level of inflammatory factor IL-6 in the plasma of mice and the liver and lung grinding fluid is obviously reduced.

The results in FIG. 5C show that TREM-2 knockout significantly reduces the levels of the inflammatory factor TNF- α in plasma and liver and lung lapping fluid.

It follows from this that: knocking out TREM-2 can reduce the generation of inflammatory factors of sepsis mice, and the result indicates that the blocking TREM-2 can achieve the effect of inhibiting sepsis inflammatory factor storm.

Example 6 Effect of knockout TREM-2 on fatty acid oxidative metabolism in septic mice

LPS-induced sepsis models were constructed using 4-6 week SPF grade female C57BL/6 mice. After the model is constructed for 12h, peripheral blood of the mouse is taken by an eyeball blood sampling method, and the mouse is centrifuged at 1800rpm for 10min to collect plasma. Meanwhile, 100mg of mouse lung and liver tissue was taken, washed once with precooled PBS, and 1ml of 5% NP-40/ddH was added₂And O, fully homogenizing the mixture by using a tissue homogenizer, then putting the homogenized mixture into a water bath kettle, slowly heating the sample to 80-100 ℃, heating for 2-5min until the solution becomes turbid, and then cooling to room temperature. This was repeated until all the triglycerides were dissolved and extracted. Triglyceride levels in plasma and liver were measured and the results are shown in figure 6A. FIG. 6A shows that WT mice have elevated serum and liver triglyceride levels, indicating reduced fatty acid oxidative breakdown, and TREM-2^-/-Triglyceride levels in the serum and liver of mice are reduced, suggesting increased fatty acid oxidation following TREM-2 knockout.

After 12h of modeling, one leaf of liver tissue was cryosectioned and stained with oil red O to assess lipid accumulation in the liver, and the results are shown in fig. 6B. FIG. 6B shows, TREM-2^-/-The accumulation of mouse liver lipid is reduced, and the knockout of TREM-2 is also suggested to improve the oxidative decomposition of fatty acid.

And (3) taking the residual liver tissue (lever) and part of lung tissue (lung), adding a protein lysate for full lysis, extracting protein, quantifying, and detecting the expression level of fatty acid oxidation related molecules by a protein immunoblotting technology, wherein the result is shown in fig. 6C. FIG. 6C results show that TREM-2 relative to WT mice^-/-The expression of fatty acid oxidation rate-limiting enzyme CPT1 and key molecules PPAR and PGC-1 participating in fatty acid oxidation in lung and liver tissues of a mouse is increased, which indicates that TREM2^-/-Increased fatty acid oxidation in mice.

After 12h of modeling, macrophages in the abdominal cavity of the mouse were taken, and the oxidation level of fatty acid of the macrophages was measured using a hippocampal cell energy metabolism analyzer, with the result shown in fig. 6D. FIG. 6D results show that TREM2 is relative to WT mice^-/-Huge abdominal cavity of mouseThe oxidation rate of phagocytic fatty acid is obviously increased, and the fact that the blocking TREM2 can improve the oxidative metabolism of the fatty acid in a mouse body is directly proved.

In summary, the following steps: the knockout of TREM-2 can improve the fatty acid oxidative metabolism of a sepsis mouse, and the blockage of TREM-2 can regulate the fatty acid oxidative metabolism disorder caused by sepsis.

Due to the reasons of different in-vivo intervention means of TREM-2, different methods for constructing sepsis models, different time phases of detection indexes, different selected cell lines and the like, the conclusion obtained by the method is opposite to that of partial research of the existing TREM-2 in sepsis. However, the invention mutually verifies the action of TREM-2 in sepsis from different aspects such as clinical samples, animal experiments, in vitro cells and the like, and adopts three sepsis models which are internationally acknowledged at present to obtain consistent conclusions, namely, the blockage of TREM-2 can improve the survival rate of sepsis mice and relieve organ damage and fatty acid oxidative metabolism disorder caused by sepsis, and the conclusions are powerfully embodied in the invention. Therefore, the invention considers that the effect of treating sepsis can be achieved by blocking TREM-2.

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Application of myeloid cell trigger receptor 2 in preparation of sepsis related functional products

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