阅读说明：本技术 一种阿尔兹海默病果蝇模型的构建方法及应用 (Construction method and application of Drosophila model of Alzheimer's disease ) 是由 孙艺昊 邱宇 徐清波 雒晶晶 薛雷 于 2021-07-23 设计创作，主要内容包括：本发明属于动物模型构建技术领域,公开了一种阿尔兹海默病果蝇模型的构建方法及应用。该阿尔兹海默病果蝇模型的构建方法,包括以下步骤：以果蝇为模式动物,在翅神经元中过表达APP基因,并使用荧光蛋白对所述翅神经元进行标记,构建得到所述阿尔兹海默病果蝇模型。经所述构建方法得到的阿尔兹海默病果蝇模型可被应用于AD发病机理研究和药物筛选,能够方便地进行活体观察,操作简单,且不会影响动物的存活情况。(The invention belongs to the technical field of animal model construction, and discloses a construction method and application of an Alzheimer disease drosophila model. The construction method of the Alzheimer disease drosophila model comprises the following steps: and (3) overexpressing an APP gene in a wing neuron by taking the drosophila as a model animal, and marking the wing neuron by using fluorescent protein to construct and obtain the model of the drosophila of the Alzheimer disease. The Drosophila model of Alzheimer's disease obtained by the construction method can be applied to research on pathogenesis of AD and drug screening, living body observation can be conveniently carried out, operation is simple, and survival conditions of animals cannot be influenced.)

1. A construction method of a drosophila model of Alzheimer's disease is characterized by comprising the following steps:

and (3) overexpressing an APP gene in a wing neuron by taking the drosophila as a model animal, and marking the wing neuron by using fluorescent protein to construct and obtain the model of the drosophila of the Alzheimer disease.

2. The method of claim 1, wherein the gene encoding the fluorescent protein is located on chromosome II and the APP gene is located on chromosome III.

3. The construction method according to claim 1, wherein the fluorescent protein is selected from one of red fluorescent protein, green fluorescent protein, yellow fluorescent protein and cyan fluorescent protein.

4. The method of claim 3, wherein the fluorescent protein is the red fluorescent protein mCD 8-mCherry.

5. The building method according to claim 4, characterized in that it comprises the steps of:

(1) the gene type is dpr-Gal4, UAS-mCD8-mCherry/SM6 a-Cyo; drosophila strain A of sb/TM6 b-Tb; and, constructing the genotype as Sp/SM6 a-Cyo; drosophila strain B of UAS-APP/TM 6B-Tb;

(2) hybridizing the drosophila strain A with the drosophila strain B to construct a gene type dpr-Gal4, UAS-mCD8-mCherry/SM6 a-Cyo; the drosophila strain of UAS-APP/TM6b-Tb is the model of the Drosophila alzeri disease.

6. The method of constructing according to claim 5, wherein the method of constructing Drosophila strain A comprises the steps of:

(A1) hybridizing a dpr-Gal4 strain drosophila with a UAS-mCD 8-mChery strain drosophila, and selecting the genotype dpr-Gal4/UAS-mCD 8-mChery from filial generations; virgins of +/- +/+;

(A2) setting the genotype as dpr-Gal4/UAS-mCD 8-mCherry; +/+ virgins with Sp/SM6 a-Cyo; the sb/TM6b-Tb line male drosophila melanogaster is hybridized, and the genotype selected from the filial generation is dpr-Gal4, UAS-mCD8-mCherry/SM6 a-Cyo; TM6b-Tb/+ Drosophila androgenic;

(A3) the genotype is dpr-Gal4, UAS-mCD8-mCherry/SM6 a-Cyo; TM6b-Tb/+ Drosophila androgens and Sp/SM6 a-Cyo; the sb/TM6b-Tb strain virgins are crossed, and the genotypes of dpr-Gal4, UAS-mCD8-mCherry/SM6a-Cyo are selected from the filial generation; the drosophila of sb/TM6b-Tb was constructed as a drosophila strain.

7. The method of claim 6, wherein the drosophila dpr-Gal4 strain is virginia virginica and the UAS-mCD8-mCherry strain drosophila male drosophila.

8. The method of claim 6, wherein said selecting in step (A2) is performed using a stereoscopic fluorescence microscope.

9. The method of constructing according to claim 5, wherein the method of constructing Drosophila strain B comprises the steps of:

(B1) Sp/SM6 a-Cyo; hybridizing virgin flies of sb/TM6b-Tb strain with male drosophila strains of UAS-APP strain, and selecting gene types of +/SM6a-Cyo from filial generations; drosophila male of UAS-APP/TM6 b-Tb;

(B2) setting the genotype as +/SM6 a-Cyo; drosophila androsaceus of UAS-APP/TM6b-Tb and Sp/SM6 a-Cyo; hybridizing the virgins of the sb/TM6b-Tb strain to obtain the genotype Sp/SM6 a-Cyo; drosophila strain of UAS-APP/TM6 b-Tb.

10. Use of the construction method according to any one of claims 1 to 9 for drug screening or pathogenesis research.

Technical Field

The invention belongs to the technical field of animal model construction, and particularly relates to a construction method and application of an Alzheimer disease drosophila model.

Background

Alzheimer's Disease (AD) is a clinically common degenerative disease of the central nervous system, and is typically characterized by progressive loss of memory and other cognitive functions that can pose serious risks to the physical and mental health of the elderly, and has become the cause of death next to heart disease, malignancy and stroke in position 4. And the alzheimer disease currently lacks an effective treatment means, so the disease becomes one of the most troublesome and urgent problems to be solved in the elderly medicine.

At present, the pathogenesis of AD is various, such as amyloid (A beta) hypothesis, tau hypothesis, mitochondrial cascade hypothesis, neuroinflammation hypothesis, free radical injury theory, cholinergic theory, calcium balance disorder theory, excitatory amino acid toxicity theory and the like, but the underlying etiology and pathogenesis of AD are not clear yet. Of most interest are the a β hypothesis: a β (amyloid protein) is a short peptide of 37-44 amino acids produced by proteolysis of APP (amyloid precursor protein), and when APP is cleaved to form a large amount of non-soluble a β 42, the aggregated a β 42 interferes with neuronal function, ultimately triggering alzheimer's disease. Abnormal expression and metabolism of APP may be closely related to the pathogenesis of AD patients.

However, the currently constructed animal model of alzheimer's disease is usually a toxicity model, which can seriously affect the survival condition of animals with the increase of age, and is not beneficial to the observation of pathological processes in vivo. Therefore, the lack of a more appropriate animal model of alzheimer's disease has become a bottleneck for restricting anti-AD drug screening and pathogenesis research.

Based on the above, the invention hopes to provide a more effective construction method of the Alzheimer's disease animal model, and lays a foundation for further developing researches on AD pathogenesis, drug screening and the like.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a construction method of the Alzheimer disease drosophila model, and the Alzheimer disease drosophila model obtained by the construction method can be applied to AD pathogenesis research and drug screening, can be used for living observation, and does not influence the survival condition of animals.

The invention provides a construction method of an Alzheimer disease drosophila model, which comprises the following steps:

and (3) overexpressing an APP gene in a wing neuron by taking the drosophila as a model animal, and marking the wing neuron by using fluorescent protein to construct and obtain the model of the drosophila of the Alzheimer disease.

Studies have shown that neurite degeneration is one of the prominent pathological features of acute nerve injury and many human neurodegenerative diseases, and part of the clinical symptoms of neurodegenerative diseases are caused by neurite degeneration. Therefore, the research on the connection between axonal degeneration and neurodegenerative diseases helps us to better understand the pathogenesis of neurodegenerative diseases and provides a new reference for the treatment of the diseases. According to the invention, the APP gene (pathogenic gene of Alzheimer disease) is over-expressed in wing neurons, and is found to cause axonal degeneration of the wing neurons, so that an axonal degeneration model is formed.

In addition, because the neurons at the wing edges of the drosophila are simple in composition and consist of mechanical sensory neurons and chemical sensory neurons, after the neurons sense external information, nerve signals can be transmitted into a central nerve-thoracic ganglion (ganglion) through axons, the axons of the neurons extend in the arch region of the wings and are converged into a bundle of nerve bundles, and the arch region has no cell body structure, so that the neuron can be used as an ideal region for observing axon change; secondly, the permeability of the wings of the drosophila is strong, so that the sub-microstructure at the edges of the wings can be observed conveniently; meanwhile, the wings of the fruit flies are convenient to pick, and the fruit flies can be directly filmed and observed. Therefore, the fluorescence protein is also marked on the basis of overexpression of the APP gene in the winged nerve of the drosophila, so that living observation is more convenient, axon degeneration of the wings of the drosophila does not influence survival of the drosophila, and change conditions of the axon of the wings of the drosophila at different ages can be continuously recorded.

Experiments show that the fruit fly model constructed by the construction method has the characteristics that the axon degeneration phenotype of the wing neurons is aggravated and the axon fragmentation degree is aggravated with the increase of age, so that the axon degeneration phenotype shows the correlation with the fruit fly age, the phenotype not only accords with the characteristics that the incidence of Alzheimer Disease (AD) is closely related to the age, but also simulates the pathological characteristics of Alzheimer disease in a fruit fly nervous system. Meanwhile, the constructed drosophila model is treated by adopting the pharmaceutical ingredients with the functions of treating and improving the Alzheimer disease, and the result shows that the axon degeneration phenotype of the drosophila model is relieved. The results show that the drosophila model of the Alzheimer disease constructed by the invention can fully simulate the pathological characteristics of the Alzheimer disease, and can be applied to the screening of anti-AD drugs and the research of pathogenic mechanisms.

Preferably, the gene encoding the fluorescent protein is located on chromosome II and the APP gene is located on chromosome III.

Preferably, the fluorescent protein is selected from one of red fluorescent protein, green fluorescent protein, yellow fluorescent protein or cyan fluorescent protein. Compared with other marking methods, the fluorescent protein marking method has better operability and good marking effect.

More preferably, the fluorescent protein is a red fluorescent protein mCD 8-mCherry. To achieve a better labeling effect, neurons at the wing edges of drosophila can be labeled with a cell membrane localized red fluorescent protein (mCD 8-mCherry).

Further preferably, the construction method comprises the following steps:

(1) the gene type is dpr-Gal4, UAS-mCD8-mCherry/SM6 a-Cyo; drosophila strain A of sb/TM6 b-Tb; and, constructing the genotype as Sp/SM6 a-Cyo; drosophila strain B of UAS-APP/TM 6B-Tb;

(2) hybridizing the fruit fly strain A with the fruit fly strain B to construct a gene type dpr-Ga l4, UAS-mCD8-mCherry/SM6 a-Cyo; the drosophila strain of UAS-APP/TM6b-Tb is the model of the Drosophila alzeri disease.

By adopting the construction method, the stably inherited model strain of the Drosophila alzeri with Alzheimer disease can be constructed

The construction method of the drosophila strain A comprises the following steps:

(A1) hybridizing a dpr-Gal4 strain drosophila with a UAS-mCD 8-mChery strain drosophila, and selecting the genotype dpr-Gal4/UAS-mCD 8-mChery from filial generations; virgins of +/- +/+;

(A2) setting the genotype as dpr-Gal4/UAS-mCD 8-mCherry; +/+ virgins with Sp/SM6 a-Cyo; the sb/TM6b-Tb line male drosophila melanogaster is hybridized, and the genotype selected from the filial generation is dpr-Gal4, UAS-mCD8-mCherry/SM6 a-Cyo; TM6b-Tb/+ Drosophila androgenic;

(A3) the genotype is dpr-Gal4, UAS-mCD8-mCherry/SM6 a-Cyo; TM6b-Tb/+ Drosophila androgens and Sp/SM6 a-Cyo; the sb/TM6b-Tb strain virgins are crossed, and the genotypes of dpr-Gal4, UAS-mCD8-mCherry/SM6a-Cyo are selected from the filial generation; the drosophila of sb/TM6b-Tb was constructed as a drosophila strain.

Further preferably, the dpr-Gal4 strain fruit flies are virgins.

Further preferably, the UAS-mCD 8-mChery strain fruit flies are male fruit flies.

Further preferably, the selection in step (a2) is performed using a stereoscopic fluorescence microscope.

The construction method of the drosophila strain B comprises the following steps:

(B1) Sp/SM6 a-Cyo; hybridizing virgin flies of sb/TM6b-Tb strain with male drosophila strains of UAS-APP strain, and selecting gene types of +/SM6a-Cyo from filial generations; drosophila male of UAS-APP/TM6 b-Tb;

(B2) setting the genotype as +/SM6 a-Cyo; drosophila androsaceus of UAS-APP/TM6b-Tb and Sp/SM6a-Cy o; hybridizing the virgins of the sb/TM6b-Tb strain to obtain the genotype Sp/SM6 a-Cyo; drosophila strain of UAS-APP/TM6 b-Tb.

The invention also provides application of the construction method of the Alzheimer disease drosophila model in drug screening or pathogenic mechanism research. The drosophila model of the Alzheimer disease constructed by the invention can fully simulate the pathological characteristics of the Alzheimer disease, can be used for in vivo observation, and can not influence the survival condition of animals, so that the drosophila model can be applied to the screening of anti-AD drugs and the research of pathogenic mechanisms.

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

according to the invention, the fruit fly overexpresses a pathogenic gene APP in the wing neurons, the wing neurons are marked by using fluorescent protein, and finally an Alzheimer disease fruit fly model capable of being stably inherited is constructed. The Alzheimer disease drosophila model can be used for conveniently and directly carrying out in-vivo imaging observation, and axon degeneration at the positions of the wing nerves of the drosophila does not influence survival of the drosophila, so that change conditions of the wing axons of the drosophila at different ages can be continuously recorded, and the Alzheimer disease drosophila model is very favorable for developing research on AD pathogenesis and screening anti-AD drugs.

Drawings

FIG. 1 shows a microscopic view of red fluorescent protein-labeled wing-edge neurons; wherein A is a schematic diagram of the distribution condition of wing edge neurons, and a main observation area is in a box; b is a picture displayed after the square frame in the A is enlarged;

FIG. 2 shows axonal degeneration in Drosophila melanogaster genotypes at different ages; wherein A-C is the degeneration condition of the axon of the drosophila melanogaster of 3 days old, D-F is the degeneration condition of the axon of the drosophila melanogaster of 15 days old, G-I is the degeneration condition of the axon of the drosophila melanogaster of 30 days old, and J is the statistical analysis result of the grade data of the axon degeneration in the drosophila melanogaster of each genotype; ns represents P > 0.05; denotes P < 0.05; p <0.0001, and the number of wings of each genotype is not less than 15;

FIG. 3 shows a Drosophila axonal degeneration assessment system; wherein a represents a complete, smooth and continuous fibrous structure of the distal neuronal axon, rated "0"; b indicates the appearance of a bead (beading) on the distal nerve axon, with a rating of "1"; c represents massive fragmentation of neurites, grade "2"; d represents the neurite presents with aggregated plaque, the axon is discontinuous, and the grade is '3'; e represents that the neurite structure is completely destroyed, and the mCD8-mCherry fluorescence signal is largely lost, and the grade is 4;

FIG. 4 shows the effect of down-regulation of β -secretase and γ -secretase on APP-induced axonal degeneration; wherein A-D is degeneration condition of axon of each genotype fruit fly of 3 days old, E-H is degeneration condition of axon of each genotype fruit fly of 15 days old, I-L is degeneration condition of axon of each genotype fruit fly of 30 days old, and M is statistical chart of axon degeneration grade condition of each genotype fruit fly in graphs A-L; ns represents P > 0.05; denotes P < 0.01; p <0.0001, and the number of wings of each genotype is not less than 15;

FIG. 5 shows the effect of apoptosis on APP-induced axonal degeneration; A-E is the axonal condition of the drosophila melanogaster with different genotypes at 3 days of age, F-J is the axonal condition of the drosophila melanogaster with different genotypes at 30 days of age, and K is the statistical analysis result of the axonal degeneration grade data of the drosophila melanogaster with different genotypes; ns represents P >0.05, represents P <0.0001, and the number of wings of each genotype is not less than 15;

FIG. 6 shows the effect of autophagy on APP-induced axonal degeneration; A-E is the change condition of the axons of the drosophila melanogaster with different genotypes at 3 days of age, F-J is the change condition of the axons of the drosophila melanogaster with different genotypes at 30 days of age, and K is the statistical analysis result of the degradation grade data of the axons of the drosophila melanogaster with different genotypes; ns represents P >0.05, represents P <0.0001, and the number of wings of each genotype is not less than 15;

FIG. 7 shows the effect of crude extract of Chinese herbs on APP-induced degeneration of drosophila pterygoid axons; wherein A-D is the change condition of the axons of each group of drosophila melanogaster with the age of 3 days, E-H is the change condition of the axons of each group of drosophila melanogaster with the age of 30 days, and I is a statistical analysis chart of the degeneration grade data of the axons of each group of drosophila melanogaster; ns denotes P > 0.05; denotes P < 0.01; denotes P < 0.001. Control represents Control group, R.S. represents added ginseng crude extract, R.C.R. represents added cistanche crude extract, and H.S.W. represents added polygonum multiflorum crude extract.

Detailed Description

In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples are given for illustration. It should be noted that the following examples are only preferred embodiments of the present invention, and the claimed protection scope is not limited thereto, and any modification, substitution, combination made without departing from the spirit and principle of the present invention are included in the protection scope of the present invention.

The starting materials, reagents or apparatuses used in the following examples are conventionally commercially available or can be obtained by conventionally known methods, unless otherwise specified.

Example 1: construction method of Drosophila model of Alzheimer's disease

The embodiment provides a method for constructing a drosophila model of alzheimer's disease, which specifically comprises the following steps:

(1) the gene type is dpr-Gal4, UAS-mCD8-mCherry/SM6 a-Cyo; drosophila strain A of sb/TM6 b-Tb; and, constructing the genotype as Sp/SM6 a-Cyo; drosophila strain B of UAS-APP/TM 6B-Tb;

(2) hybridizing the drosophila strain A with the drosophila strain B to construct a gene type dpr-Gal4, U AS-mCD8-mCherry/SM6 a-Cyo; the drosophila strain of UAS-APP/TM6b-Tb is the model of Drosophila alzeri disease.

The construction method of the drosophila strain A comprises the following steps:

(A1) hybridizing virgins of a dpr-Gal4 strain (purchased from the American Blumeton Drosophila strain center, number: 25083) with male drosophila of a UAS-mCD8-mCherry strain (obtained from the Central academy of sciences biochemistry crossover research center), and selecting the genotype dpr-Gal4/UAS-mCD8-mCherry from filial generations; virgins of +/- +/+;

(A2) the genotype is dpr-Gal4/UAS-mCD 8-mCherry; +/+ virgins with Sp/SM6 a-Cyo; s b/TM6b-Tb strain male drosophila melanogaster is hybridized, and the gene type dpr-Ga l4, UAS-mCD8-mCherry/SM6a-Cyo are selected from offspring by adopting a stereoscopic fluorescence microscope; TM6b-Tb/+ Drosophila androgenic;

(A3) the gene type is dpr-Gal4, UAS-mCD8-mCherry/SM6 a-Cyo; TM6b-Tb/+ Drosophila androgens and Sp/SM6 a-Cyo; the sb/TM6b-Tb strain virgins are crossed, and the genotypes of dpr-Ga l4, UAS-mCD8-mCherry/SM6a-Cyo are selected from the filial generation; the drosophila of sb/TM6b-Tb was constructed as a drosophila strain.

The construction method of the drosophila strain B comprises the following steps:

(B1) Sp/SM6 a-Cyo; virgins of the sb/TM6b-Tb line (obtained from the institute of Life sciences and technology, university of Tongji) were crossed with male drosophila lines of the UAS-APP line (obtained from the molecular biology center, university of Heidelberg, Germany), selecting from the progeny the genotype +/SM6 a-Cyo; drosophila male of UAS-APP/TM6 b-Tb;

(B2) setting the genotype as +/SM6 a-Cyo; drosophila androsaceus of UAS-APP/TM6b-Tb and Sp/SM6 a-Cyo; hybridizing the virgins of the sb/TM6b-Tb strain to obtain the genotype Sp/SM6 a-Cyo; drosophila strain of UAS-APP/TM6 b-Tb.

Example 2: observation of the winged nerve of the Drosophila model of Alzheimer's disease

The observation is carried out aiming at the drosophila model of the Alzheimer disease constructed in the embodiment 1, and the steps are as follows:

1. in CO₂Anaesthetizing the fruit fly to be observed (the Alzheimer disease fruit fly model constructed in the embodiment 1) on an air plate, fixing the body of the fruit fly by using the dissecting forceps of the left hand, quickly and accurately picking the Wing of the fruit fly by using the dissecting forceps of the right hand, and immediately placing the picked Wing in the Wing Wash Buffer (WWB) solution;

preparation of WWB solution: mixing 1 XPBS solution, formaldehyde reagent and Triton X-100 solution to make the final concentration of formaldehyde be 4% and the final concentration of Triton X-100 be 0.2%; i.e., 900mL of PBS solution +100mL of formaldehyde reagent +2mL of Triton X-100 reagent. WWB solutions can be stored at-20 ℃ for one month, but their fixation efficiency decreases with time;

2. taking a clean glass slide, and dripping a drop of a proper amount of ethanol-glycerol mixed solution (glycerol: ethanol is 3: 1, and the mixed solution is used immediately);

3. gently clamping one side of the wing without neurons by using dissecting forceps, gently sucking the wing and liquid on the forceps by using absorbent paper, then placing the wing in an ethanol-glycerol buffer solution, and paying attention to the fact that the ventral side of the wing is placed upwards.

4. The cover glass is slowly covered, and then shooting imaging can be carried out for observation (the neuron at the edge of the wing needs to be shot by using a high power lens of more than 40 times, and each glass slide is provided with 6 wings at most).

FIG. 1 is a microscopic view of a red fluorescent protein labeled wing edge neuron, wherein A is a schematic representation of the distribution of the wing edge neuron and the main observation area is within the box; and B is a picture displayed after the square frame in the A is enlarged.

To explore the change in axons in the wing arch region of the drosophila model of alzheimer's disease over time. The Drosophila model of Alzheimer's disease was set as an experimental group (APP group), and w was¹¹¹⁸The group (wild Drosophila melanogaster, obtained from the college of Life sciences and technology of Tongji university) was used as a blank control group, and the group (the experimental group and the control group both use a dpr-Gal4 system to mark red fluorescent protein mCD 8-mChery on Drosophila melanogaster neurons) used as a negative control group, wherein the group (the Dcr2 protein is a member of double-stranded RNA specific endonuclease RNase III family, is usually expressed as a control protein and is purchased from the American Blugington Drosophila strain center, and the number is 24651) used for over-expressing Dcr2 (the Dcr2 protein is a member of double-stranded RNA specific endonuclease RNase III family), and the axon change conditions of Drosophila melanogaster of different age groups are observed.

The results are shown in FIG. 2: at 3 days of age, the axons were smooth and flat, fibrous, both in control drosophila (a-B in fig. 2) and in APP-overexpressing drosophila (C in fig. 2); at 15 days, the axons of the drosophila melanogaster control group (D-E in FIG. 2) remained smooth and flat, while the axons of the drosophila APP group (F in FIG. 2) showed many beaded spots and axon fragments, indicating that the axons had degenerated; by day 30, the control drosophila (G-H in fig. 2) had intact axonal structures with very few fragments; while the axon integrity of the APP drosophila (I in FIG. 2) is destroyed, discrete large plaques appear, the loss of fluorescence signal is weakened, and axon degeneration is aggravated. The results show that the overexpression of the APP gene can cause axonal degeneration on the neuroaxons of the drosophila wings, and the phenotype of the axonal degeneration is related to the drosophila age, so that the phenotype not only accords with the characteristics of AD morbidity and age close correlation, but also simulates the pathological characteristics of AD in the drosophila nervous system. In FIG. 2, J is a statistical analysis of data on the grade of axonal degeneration of drosophila melanogaster of different genotypes, and for the grade evaluation of axonal degeneration, as shown in FIG. 3, axonal degeneration is divided into 5 grades, which are sequentially represented by 0 to 4, wherein A represents that the axon of the distal neuron presents a complete, smooth and continuous fibrous structure, and the grade is "0"; b indicates the appearance of a bead (beading) on the distal nerve axon, with a rating of "1"; c represents massive fragmentation of neurites, grade "2"; d represents the neurite presents with aggregated plaque, the axon is discontinuous, and the grade is '3'; e indicates that the neurite structure was completely destroyed and that the mCD8-mCherry fluorescence signal was largely lost, rated "4". The results show that the overexpression of the APP gene can cause axonal degeneration on the neural axons of the drosophila wings, the phenotype of the axonal degeneration is related to the drosophila age, the phenotype meets the characteristics that the AD onset is closely related to the age, and the pathological characteristics of AD are simulated in the drosophila nervous system.

Example 3: verification experiment of Drosophila model of Alzheimer's disease

The Alzheimer disease drosophila model constructed in the example 1 is taken as an experimental object, and the specific dependence of the drosophila model on the APP gene is verified. Since the APP amyloid cleavage pathway produces pathogenic A beta process, needs to go through key cleavage enzyme beta-secretase and gamma-secretase. It has been concluded that interference of these two enzymes by RNAi interferes with the production of a β, thus alleviating the senile dementia caused by APP. This example down-regulates the expression of β -secretase (down-regulated Bace gene) and γ -secretase (down-regulated Psn gene), respectively, while over-expressing APP. The results of the experiment are shown in FIG. 4: no axon degeneration phenotype was observed at the axons for 3-day-old genotyped Drosophila flies (A-D in FIG. 4); at day 15 and day 30, with APP groups (E and I in fig. 4) and APP; compared with Dcr2 groups (F and J in FIG. 4), downregulation of Bace (G and K in FIG. 4) and Psn (H and L in FIG. 4) can both significantly inhibit axonal degeneration. The results show that the down regulation of beta-secretase and gamma-secretase can effectively inhibit the axonal degeneration phenotype caused by APP, and the constructed model of the drosophila alzeheimer disease exclusively depending on APP is proved.

Example 4: application of Alzheimer disease fruit fly model in pathogenesis research

1. Investigating the Effect of apoptosis on APP-induced axonal degeneration

Axonal degeneration is the process by which axons actively destroy themselves, and what mechanism mediates the axonal degeneration caused by APP remains to be studied further. In order to investigate whether axon degeneration caused by overexpression of APP depends on apoptosis, the experiment subject is the drosophila alzheimer disease model constructed in example 1, and measures for inhibiting apoptosis such as overexpression of P35, DIAP1 or knockdown of Dcp1 are respectively taken. The results of the experiment are shown in FIG. 5: A-E is the axonal condition of the drosophila melanogaster with different genotypes at 3 days of age, F-J is the axonal condition of the drosophila melanogaster with different genotypes at 30 days of age, and K is the statistical analysis of the axonal degeneration grade data of the drosophila melanogaster with different genotypes. The results show that overexpression of P35, DIAP1 or downregulation of Dcp1 inhibits apoptosis, fails to reduce the axonal degeneration phenotype caused by APP, and indicates that apoptosis is not involved in axonal degeneration caused by APP.

2. Investigating the influence of autophagy on APP-induced axonal degeneration

The pathogenesis of some common neurodegenerative diseases is closely related to autophagy. The body can clear damaged, degenerating, senescent, and dysfunctional cells by autophagy. It has been reported that autophagosomal accumulation is observed in brain slices from AD patients, suggesting an abnormality in the autophagy process in the brain of AD patients, which may be caused by increased autophagy initiation or decreased clearance of lysosomes. Autophagy abnormalities were also observed in AD mouse models. However, no determination is made as to what role autophagy plays in AD at present, and studies show that autophagy is reduced in the early stage of AD; autophagy activating factors are, in turn, up-regulated in the brain of AD patients. Further studies have shown that overexpression of APP in both the brain and ocular adult discs of drosophila larvae leads to a significant increase in autophagic vesicles, and that blocking autophagy inhibits learning deficit caused by overexpression of APP.

In order to investigate whether the axonal degeneration caused by APP is related to autophagy, in this example, the alzheimer drosophila model constructed in example 1 is used as an experimental subject, and the key genes Atg7 and Atg12 in the autophagy process are respectively down-regulated to inhibit autophagy. The experimental results are shown in fig. 6: A-E in FIG. 6 are the change of the axons of the drosophila melanogaster with different genotypes at 3 days of age, F-J in FIG. 6 are the change of the axons of the drosophila melanogaster with different genotypes at 30 days of age, and K in FIG. 6 is the result of statistical analysis of the degradation grade data of the axons of the drosophila melanogaster with different genotypes. The results show that the down-regulation of autophagy can well inhibit the axonal degeneration phenotype caused by APP, and that autophagy participates in the axonal degeneration caused by APP.

Example 5: application of Alzheimer disease drosophila model in anti-AD drug screening

In order to prove that the drosophila model of alzheimer disease constructed in example 1 indeed has the effect of screening anti-AD drugs, this example screens 3 kinds of crude extracts of traditional Chinese medicine reported to have certain curative effect on treatment of senile dementia: ginseng, cistanche deserticola and polygonum multiflorum; and preparing a corresponding culture medium from the crude extract (liquid medicine) according to the proportion shown in the following table 1, and then culturing the model of the drosophila alzheimers disease.

TABLE 1 formulation of Drosophila culture Medium

1. Ginseng: is dried root of perennial herb of Panax of Araliaceae, and has effects of tranquilizing mind and improving intelligence. Researches find that the ginseng can effectively regulate the excitability of the central nervous system, promote the synthesis and the release of acetylcholine in the brain and promote the content of dopamine and epinephrine in the brain. The ginsenoside extracted from ginseng can effectively improve the spontaneous activity and the exploration behavior of Abeta rats in a new environment and enhance the learning and memory ability.

2. Cistanche deserticola: cistanche salsa is a parasitic plant parasitic on the root of the haloxylon ammodendron in desert, and absorbs nutrients and water from the host. The ginseng has good reputation of desert ginseng, has high medicinal value and is a traditional famous and precious traditional Chinese medicine. Researches find that total cistanchis glycosides extracted from cistanche deserticola have certain improvement effect on learning and memory ability of AD model mice, and simultaneously can improve the activity of SOD in brain tissues of the mice, reduce the generation of lipid peroxides and finally achieve the anti-aging effect.

3. Polygonum multiflorum: is perennial winding liana of Polygonum of Polygonaceae, and has thick root tuber and black brown color. The fleece-flower root has the efficacies of tonifying kidney and blood, blackening hair and strengthening tendons. The polygonum multiflorum can inhibit apoptosis of hippocampus cells of AD rats and activity of monoamine oxidase-B in brain tissues of AD mice, so that damage of free radicals to organisms is eliminated, and aging is further relieved. The stilbene glucoside found in Polygonum multiflorum has certain effect on preventing and treating neurodegenerative diseases such as Alzheimer disease.

After the drosophila alzheimer's disease model constructed in example 1 was treated with the above culture medium, the experimental results are shown in fig. 7: in each group of 3-day-old Drosophila, the wing axons (A-D in FIG. 7) were smooth and fibrous; at day 30, overexpression of APP (E in FIG. 7) induced axonal degeneration, while the crude extract of Ginseng radix (F in FIG. 7), the crude extract of Cistanchis herba (G in FIG. 7) and the crude extract of Polygoni Multiflori radix (H in FIG. 7) all rescued the axonal degeneration phenotype well at day 30 due to overexpression of APP. The results show that after the Drosophila model with Alzheimer disease is used, the symptoms of axonal degeneration can be improved, and the model is successfully and effectively constructed.

The embodiments of the present application have been described in detail with reference to the drawings, but the present application is not limited to the embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present application. Furthermore, the embodiments and features of the embodiments of the present application may be combined with each other without conflict.