CN115486415A - Establishing method and application of bee Parkinson model - Google Patents
Establishing method and application of bee Parkinson model Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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Abstract
The invention discloses a method for establishing a bee Parkinson model, which comprises the following steps: animal selection: selecting mature bees as objects for establishing a bee Parkinson model; preparing a rotenone sucrose pollen solution: dissolving rotenone in dimethyl sulfoxide, and adding sucrose pollen solution to prepare rotenone sucrose pollen solution; model establishment: feeding the mature bee with rotenone sucrose pollen solution for more than 3 days to obtain a bee Parkinson model; the method of the invention has simple operation, low cost and short molding period, and the established bee Parkinson model can be used for screening drugs for preventing or/and treating Parkinson-related diseases.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for establishing a bee Parkinson model and application thereof.
Background
Parkinson's Disease (PD) is a well-known common neurodegenerative disease with a global prevalence of 0.1% to 0.2%, symptoms including motor dysfunction and non-motor symptoms. Motor dysfunction is a key feature of PD and is clinically manifested by bradykinesia, resting tremor, rigidity, and postural instability, and these motor deficits are thought to be caused by degeneration of dopaminergic neurons of the brain. Whereas non-motor symptoms are related to common gastrointestinal dysfunction such as dysphagia, gastric emptying disorders and dyschezia.
Rotenone is a naturally occurring pesticide, extracted from the roots of plants of the genera Dioscorea and derris. In the 1980 s, heikkila et al used rotenone for the first time for PD studies, which reproduced some of the symptoms of clinical PD in a rat model. At present, the method for inducing PD model by rotenone is widely used in rodent, caenorhabditis elegans, drosophila, zebrafish and other animals.
The honeybee is used as a novel model animal, has the advantages of low cost, simple operation, short model building period, high flux and the like, and is particularly suitable for large-scale experimental research. In addition, bees have organs homologous or similar to humans, such as the brain, fat bodies, oocytes, gastrointestinal tract and circulatory system, which provides natural advantages for the use of bees in the construction of animal models of human diseases, as well as other simple model organisms that have been widely used in the study of human diseases. The existing various Parkinson animal models have the advantages, but part of models have higher cost, long establishment period and unstable model effect, so that the application of the models and the research on the disease mechanism of Parkinson are limited.
Disclosure of Invention
In order to overcome the defects in the prior art and reduce the cost of model establishment, the invention provides a stable and efficient establishing method of a bee Parkinson model, which can be used for screening medicines for preventing or treating Parkinson-related diseases and can be applied to the research of specific mechanisms of the Parkinson-related diseases.
The establishing method of the bee Parkinson model comprises the following steps:
animal selection: selecting mature bees as objects for establishing a bee Parkinson model; the mature bees are 2 to 15 days old, the unified type is ensured as much as possible when the bees are selected, and the bees with too small or too large body types are removed;
preparing a rotenone sucrose pollen solution: firstly, placing pollen into a sucrose solution with the mass volume concentration of 40-60% according to the proportion that the adding amount of the pollen is 25-50% of the volume of the sucrose solution, and after vortex mixing, adding a rotenone solution dissolved in dimethyl sulfoxide into a prepared sucrose pollen solution to prepare a rotenone sucrose pollen solution; adding a proper amount of pollen to ensure the feed intake of bees in the future so as to ensure the establishment of a bee Parkinson model, wherein sucrose is a main carbon source for bee diet, and the concentration of rotenone in the rotenone sucrose pollen solution is 50-250 μmol/L; the inflammation degree of the model is adjusted by changing the concentration of the rotenone, and the specific disease expression is more obvious along with the increase of the concentration of the rotenone. However, the death rate of bees is increased due to the induction of the rotenone with high concentration, so that the operable test period of the Parkinson model is shortened, and in order to avoid the insufficient survival rate of the bees and ensure the obvious Parkinson symptoms of the model, the establishment of the bee Parkinson model by using the rotenone with the concentration of 100 mu M is recommended.
Model establishment: and feeding the mature bee with the rotenone sucrose pollen solution for more than 3 days to establish a bee Parkinson model.
The sucrose solution is filtered and sterilized by a filter membrane of 0.22 mu m, so that the influence of opportunistic pathogens possibly existing in the environment is eliminated;
the pollen also uses aseptic pollen to eliminate the influence of opportunistic pathogens possibly existing in the environment, and the sterilization conditions are as follows: sterilizing for 3 to 5 hours by using an electron beam with the strength of 20 to 40kGy.
The mature bees are 2 to 15 days old bees, the bee in the application refers to the general name of other insects except ant insects in hymenoptera, the types of the bees in the application are not limited, for example, the bee product belongs to the family Apidae, the family Vespidae, the family Apidae, the genus Apis, the genus bumble bee, the genus Apis, the genus Ceriporia, the genus Apis, and the genus Apis.
The mature bee is a bee of Apidae, and is bee or bumble bee.
The 9 insects of genus Apis of family Apidae are collectively referred to as bees, and are typically characterized by collecting nectar and pollen and producing the nectar into honey, wherein the most common are Apis cerana of Apis cerana, apis mellifera of Apis cerana, etc., and additionally, apis mellifera, apis viridans, etc. The bee species in the present application is not limited, but preferably is a bee species that is easy to purchase and obtain.
More than 500 social insects of the genus bumblebee of the family Apidae are collectively called bumblebee, and are typically characterized by being thick and thick in body, densely covered by black, yellow or long hair with various colors, widely distributed around the world except Antarctica and Atlanta, living in camphol, having a swarm structure identical to that of bees, eating pollen and nectar of flowering plants, and mainly including heavy yellow bumblebee, short-headed bumblebee, and patterned bumblebee. The kind of bumble bees in the present application is not limited, but is preferably a bumble bee kind which is easy to purchase and obtain.
The process of obtaining the mature bees by breeding in the invention comprises the following steps: selecting a honeycomb spleen, placing the honeycomb in an incubator with the temperature of 25-30 ℃ and the humidity of 50%, culturing overnight by using a sucrose solution, observing the eclosion condition of bee pupae, and culturing to obtain the eclosion bee. And (3) continuously culturing the eclosion bees for 1 to 13 days, feeding sterile sucrose and sterile pollen solution in the culture process, and eliminating the influence of opportunistic pathogenic bacteria possibly existing in the environment to obtain mature bees.
The preparation process of the sterile pollen comprises the following steps: subpackaging the pollen into a self-sealing bag, strictly sealing the self-sealing bag, and sterilizing for 3 to 5 hours by using electron beams with the strength of 20 to 40kGy to obtain sterile pollen.
The invention also aims to apply the bee Parkinson model constructed by the method to screening of drugs for preventing or/and treating Parkinson-related diseases or to research of Parkinson-related disease mechanisms.
The medicine comprises a chemical preparation and a biological preparation.
The invention has the advantages and technical effects that:
the method takes the mature bees as the object for establishing the bee Parkinson model, can efficiently initiate the bee nerve inflammation, reduces the dopaminergic neurons of the bee brain, reduces the expression quantity of the dopaminergic neuron marker gene, further causes the motor behavior disorder, and simultaneously reproduces the symptom of the gastrointestinal dysfunction accompanying the Parkinson disease.
Compared with other common animal models in the prior art, the model is simple and convenient to operate, low in cost, short in modeling period, remarkable in Parkinson disease sign and adjustable in disease degree, is suitable for large-scale experiments of the Parkinson model, can be used for screening new methods or new drugs for preventing or treating Parkinson-related diseases, and can be applied to exploring specific mechanisms of the Parkinson-related diseases.
Drawings
FIG. 1 shows the results of the effect of rotenone on survival curves and body weights of bumblebee Parkinson models, wherein A is the survival rate and B is the body weight;
FIG. 2 is the result of the influence of rotenone on the flying motion ability of bumblebee Parkinson model;
FIG. 3 is a statistical result of intestinal tract total intestine length of a rotenone-induced bumblebee Parkinson model according to the invention, wherein A is a statistical histogram, B is a control group measurement object, and C is a rotenone group measurement object;
FIG. 4 is a graph showing the HE staining results of intestinal sections of a rotenone-induced bumblebee Parkinson model according to the present invention;
FIG. 5 is a graph of the brain dopaminergic neuron immunostaining of the rotenone-induced bumblebee Parkinson model of the present invention, wherein DAPI is the result of the fluorescent staining of the cell nucleus, TH is the result of the fluorescent staining of the dopaminergic neuron, and Merge is the result of the fluorescent staining combining the two;
FIG. 6 shows the expression of dopaminergic neuron markers in bumblebee Parkinson model brain induced by rotenone;
FIG. 7 is a single cell transcriptome profile using a t-SNE dimension reduction method showing brain data of bumblebee merged with the Parkinson model and a normal control bumblebee;
FIG. 8 is the results of subpopulation grouping of Glaa cell populations using the t-SNE dimension reduction method;
FIG. 9 shows the results of subpopulation grouping of KC cell populations using the t-SNE dimension reduction method;
FIG. 10 shows the results of subpopulation grouping of OLC cell populations using the t-SNE dimension reduction method;
FIG. 11 shows the results of subpopulation grouping of OPN cell populations using the t-SNE dimension reduction method;
FIG. 12 shows the results of KEGG pathway enrichment analysis of upregulated genes after treatment with rotenone from AST2 cell subset;
FIG. 13 shows the result of KEGG pathway enrichment analysis of upregulated genes after treatment of the mKC cell subset rotenone;
FIG. 14 shows the result of KEGG pathway enrichment analysis of upregulated genes after treatment with rotenone in lKC cell subset.
Detailed Description
The following description of the exemplary embodiments of the present application, including various details of the embodiments of the present application to assist in understanding, should be taken as exemplary only. Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope and spirit of the present application. Also, descriptions of well-known functions and constructions are omitted in the following description for clarity and conciseness.
Example 1: establishing Parkinson model by taking bumblebee as example
1. Preparation of early stage of experiment
Weighing 500g of sucrose, using ultrapure water to fix the volume to 1L to obtain a sucrose solution with the mass volume concentration of 50%, and taking a 0.22 mu m filter membrane for filtration and sterilization to obtain a sterile sucrose solution required by an experiment. Subpackaging purchased pollen (rape pollen in Qinghai province) into a self-sealing bag, ensuring strict sealing, sterilizing for 4h by using an electron beam with the strength of 30kGy to obtain sterile pollen, adding sterile pollen with the volume of 25% into a 50% sterile sucrose solution, and performing vortex mixing to obtain the sterile sucrose pollen solution.
Dissolving rotenone (product of Meclin company) in dimethyl sulfoxide, adding prepared sterile sucrose pollen solution, and preparing rotenone sucrose pollen solution with rotenone concentration of 100 μmol/L.
2. Selection and culture of experimental bumble bees
And (3) observing the maturation condition of the bee pupae, selecting a proper honeycomb spleen, putting the honeycomb spleen into a clean plastic square box, and inserting a 2mL centrifuge tube (with a perforated tube wall) filled with a sterile sucrose solution into the side or the upper part of the box body. And (3) putting the square box containing the bee pupae into an incubator with the temperature of 25 to 30 ℃ and the humidity of 50% for overnight culture.
Observing the bee emergence condition of the honeycomb spleen on the second day, gently taking out the bees which are emerged and come out of the honeycomb, subpackaging the bees into disposable transparent culture cups, feeding 50% sterile sucrose solution to each new cup of bumble bees, continuously placing the bumble bees in a constant-temperature culture box for 1-2 days, and performing Parkinson induction after the bumble bees adapt to a new environment.
3. Animal grouping and Experimental design
Selecting mature bumblebees as objects for establishing a bumblebee Parkinson model, and setting four groups: a sampling group, a survival curve testing group, a weight testing group and a flight ability testing group;
randomly selecting 48 normal bombus as a sampling group, and dividing the sampling group into 2 groups: a control group and a rotenone group, wherein each group contains 24 bumblebees for experimental sampling; randomly selecting 48 normal bumblebees as a survival curve test group, dividing the survival curve test group into a control group and a rotenone group, and observing the survival condition of 24 bumblebees in each group; randomly selecting 48 normal bumblebees as a weight test group, dividing the weight test group into a control group and a rotenone group, wherein each group comprises 24 bumblebees for observing the weight change condition; randomly selecting 100 normal bumblebees as a flight capability test group, dividing the test group into a control group and a rotenone group, wherein each group comprises 50 bumblebees, and is used for testing the flight motion capability of the bumblebees.
In the survival curve test group, the weight test group and the sampling group, the control group freely takes the conventional sterile sucrose pollen solution, and the rotenone group freely takes the rotenone sucrose pollen solution with the concentration of 100 mu mol/L; the flight ability test group freely takes conventional sterile sucrose pollen solution in the first 7 days to ensure the good development of the bumblebees in the early growth stage, the control group freely takes conventional sterile sucrose pollen solution in the 7 th day, and the rotenone group freely takes rotenone sucrose pollen solution with the concentration of 100 mu mol/L.
The bumblebee takes the rotenone sucrose pollen solution prepared in advance and each control solution for more than 3 days in a free feeding mode according to the grouping design.
Example 2: survival curves and weight detection
After the survival curve test group and the weight test group are divided into groups, the bumblebees take the rotenone sucrose pollen solution and the sterile sucrose pollen solution which are prepared in advance in a free feeding mode, and the survival conditions and the weight changes of the bumblebees in each group are observed and recorded every day; bumblebees which are possibly caused by development defects or artificial physical injuries die before rotenone intervention is carried out, and the data are eliminated and are not taken as final statistical data;
the results are shown in figure 1, when 100 mu mol/L of rotenone is added into the diet, the bumblebee begins to die at the 2 nd day, the survival rate at the 5 th day is less than 30%, which indicates that the rotenone induction can seriously affect the survival capability of the bumblebee, meanwhile, the rotenone can also cause the weight reduction of the bumblebee, and the weight of the bumblebee fed with the rotenone is obviously lower than that of the control group at the 2 nd day.
Example 3: flight motion capability test
The flight ability test group carries out flight ability test on the rotenone treatment group and the control group on a flight mill instrument respectively on 3 rd to 5 th days after diet change. The bees are tied to the cantilever of the flying mill, and the surrounding type rotary flying is realized by utilizing the own power. When the flying cantilever rotates, the magnetic sensor transmits voltage pulses, labView software at a computer end records voltage pulse signals, and then the flying time, the flying distance and the flying speed of each rotation and the whole flying process of bees are calculated through Microsoft Excel;
the results are shown in fig. 2, the mean flying speed of bumblebees in 100 μmol/L rotenone group is significantly lower than that in the control group, which shows that rotenone significantly reduces the flying motor ability of bumblebees and reproduces the motor disorder symptoms in parkinson's disease.
Example 4: intestinal experiment, brain immunofluorescence staining and real-time fluorescence quantitative PCR of bumblebee
Sampling the control group and rotenone group of the sampling group from 3 days to 5 days after eating (according to the requirement on the disease severity, selecting sampling time points on the premise of good survival condition of the bumblebee), dissecting a complete bumblebee intestinal tract by using forceps and measuring the length; meanwhile, collecting partial complete intestines with complete forms, fixing the intestines in a 4w/v% paraformaldehyde solution, and using the intestines for subsequent HE staining and pathological observation. Dissecting the brains of the bumblebees, taking out a part of the brains completely, and fixing the brains in 4w/v% paraformaldehyde solution for subsequent immunofluorescence staining of the brains; and freezing the other part of brain with liquid nitrogen immediately after removing glands and melanin, and storing at-80 ℃ for subsequent real-time fluorescence quantitative PCR (qPCR) analysis of related genes and single cell transcriptome sequencing.
1. Intestinal length, intestinal HE staining and pathology observation
1.1 when 100. Mu. Mol/L rotenone was added to the diet, the total intestinal length of bumblebees was significantly lower than that of the control group, see FIG. 3; one of the characteristics of the bombus parkinson induced by rotenone can cause the intestinal tract to be shortened, thereby influencing the normal function of the intestinal tract;
1.2 fixing the whole intestine with complete shape in a paraformaldehyde solution with mass volume concentration of 4%, and then sending the fixed intestinal tissue to Wuhan Seville Biotechnology limited company for subsequent experimental operations such as paraffin embedding, slicing, dyeing and the like; after the company returns to the tissue section, pathological observation is carried out under a microscope, and an Image is shot by combining Image Viewer software.
The transverse HE staining results of the ileum and the midgut of the bumblebee intestinal tract are shown in figure 4, and the results show that the influence of the rotenone treatment on the ileum is not obvious, but the phenomenon of severe intestinal wall mucous membrane thickening is caused at the midgut, and the negative influence of the rotenone treatment on the length of the bumblebee intestinal tract and the intestinal barrier is similar to the clinical gastrointestinal dysfunction symptom of the Parkinson disease.
2. Immunofluorescent staining of the brain
The method comprises the steps of sending a bumblebee brain sample fixed in paraformaldehyde solution with the mass volume concentration of 4% to Wuhan Severe Biotechnology limited company, preparing a paraffin section by the company, carrying out subsequent operations of antigen repair, circled serum blocking, antibody addition, nucleus staining, tissue autofluorescence quenching, mounting and the like, and finally observing and collecting the section under a fluorescence microscope to obtain an image.
The result is shown in figure 5, and the immunofluorescence staining result of the dopaminergic neuron of the bombus cerebrum shows that the number of dopaminergic neurons in the bombus cerebrum is reduced (TH marked neurons) after 100 mu mol/L rotenone treatment, a plurality of main neuron clusters are destroyed, which indicates that the construction of the Parkinson model is successful, and the key pathological characteristics of the Parkinson disease are reproduced.
3. Real-time fluorescent quantitative PCR (qPCR)
FastPure cell/tissue Total RNA isolation kit V2 (Nanjing Novox Co.) was used for extraction of total RNA from individual brain samples and reverse transcription of RNA was performed using HiScript III RT SuperMix for qPCR (+ gDNAwiper) reagents (Nanjing Novox Co.). Relative gene expression was measured in a fluorescent quantitative PCR instrument using a ChamQ. RTM. Universal SYBR qPCR Master Mix (Nanjing Novodka). In order to detect the transcript of the dopaminergic neuron marker gene, the invention designs a related primer: TH gene (LOC 100646624), forward primer: 5 'CGATCTTTTGGGCTTGAAGAG-3', reverse primer: 5' AGTTTCAAAGCGAGCATCGT-; actin gene (LOC 100648239, internal reference), forward primer: 5 'GATGGATGGTCCAGACTCGT-3', reverse primer: 5 'GAATCGCTGACAGAATGCAA-3'; the above primers were all synthesized by Biotechnology engineering (Shanghai) GmbH, and the TH gene was standardized using actin gene as an internal reference to 2 -ΔΔCT The method quantifies the relative expression of the target gene mRNA.
The result is shown in figure 6, qPCR is used for further verifying the damage condition of the dopaminergic neuron, the result shows that the marker gene TH of the dopaminergic neuron is obviously reduced compared with a control group after 100 mu M rotenone treatment, the marker gene TH is consistent with the immunofluorescence staining result, the rotenone can damage the dopaminergic neuron of the bumble bee brain through further verification, and the success of modeling is judged.
Example 5: brain single cell transcriptome sequencing clustering analysis
Freezing and storing the bombus brain sample without glands and melanin in liquid nitrogen, mailing dry ice to Qingdao Hua Dagenen research institute for 10X Genomics single cell transcriptome sequencing, separating out single cells based on a microfluidic technology, carrying out efficient reverse transcription amplification on trace mRNA in the single cells, and then carrying out illumina high-throughput sequencing; in single cell transcriptome sequencing analysis, a data matrix of gene expression (DGE) obtained by sequencing is first screened to remove nuclei with less than 200 or more than 2,000 expressed genes, or nuclei with a high UMI count ratio (> 1%) from mitochondrial genes. Then, seurat (v.4.0) is used for normalization, integration, dimensionality reduction, clustering, visualization and marker gene analysis in an R (v.4.0) environment to obtain a bumblebee brain single cell level transcription profile.
The gene expression matrix is subjected to normalization, integration, dimensionality reduction, clustering, visualization and marker gene analysis in an R (v.4.0) environment through Seurat (v.4.0), and then the obtained bumblebee brain atlas is clustered. 32418 cells of bumblebee brain can be clustered into 5 Cell types with different gene expression patterns according to known various Cell type marker genes, which are respectively glial cells (Glia), kenyan Cells (KC), visual leaf cells (OLC), olfactory Projection Neurons (OPN), other Neurons (Neurons, N), and the result of a clustering map obtained by using a dimension reduction method of t-SNE (t-partitioned stored Neighbor Embedding), as shown in fig. 7. By further performing subpopulation analysis on the cell populations such as Glia, KC, OLC, etc., glia can be further divided into two types of astrocytes (AST 1, AST 2), cortical Glia (COR), ganglion-encapsulated glial cells (ENS), outermost glial cells (PER) of the nervous system, and second outer glial cells (subcor) at a specific resolution, as shown in fig. 8; KCs can be further divided into small kenyang cells (sKC), medium kenyang cells (mKC), large kenyang cells (lKC), and two types of type ii kenyang cells (IIKC 1, IIKC 2), see fig. 9; OLCs can be further divided into six sub-populations of OLC cells that cannot be fully identified (OLC 1, OLC2, OLC3, OLC4, OLC5, OLC 6), see FIG. 10; the OPN cell population was not further grouped due to the limited number of cells, as shown in FIG. 11. Then, statistically significant differential genes were screened for pathway enrichment between rotenone-treated and control groups for each cell type subpopulation, and finally, it was found that a large number of pathways for neurodegenerative diseases were significantly up-regulated after rotenone treatment, including parkinson's disease pathways, in AST2 subpopulation cells of gia, mKC of KC, and lKC subpopulation cells, compared to control groups, as seen in fig. 12-14. This suggests that the mechanism by which rotenone causes parkinsonian symptoms in bumble bees is likely to be by affecting astrocytes in the brain glial cells and the meso-and large-sized kenyan cells in the body of the brain mushroom. Research in recent years has found that astrocytes have strong correlation with genes related to Parkinson's disease, which shows that the bumblebee Parkinson model of the invention is successfully constructed and has great potential for further researching the disease mechanism of Parkinson at the single cell level. The Kenyang cells refer to internal neurons of insect brain mushroom bodies, and are important for olfactory learning and memory of insects, the results of the invention show that Parkinson's disease can affect the Kenyang cells of bumblebee brain, and further can affect olfactory and memory functions, which is consistent with the current research conclusion related to Parkinson's disease, parkinson's disease people can have symptoms of olfactory decline in early stage, and the decline of memory and cognitive ability is also one of the main symptoms of Parkinson's disease. However, in the existing research on the animal model of Parkinson's disease, particularly in the animal model of insects, such as the drosophila Parkinson's model with a mature technology, the correlation between Parkinson's disease and Kenyang cells has not been reported in detail, so that the invention provides a new diagnosis index for the animal model of Parkinson's disease and a new research direction for researching the molecular mechanism of the single cell level of Parkinson's disease.
The results show that the establishing method of the bee Parkinson model is applicable to bees, particularly bees in the family Apidae, such as bumblebees and the like, and the method successfully reproduces a plurality of clinical manifestations of the Parkinson disease. But for different bee types, the respective specific experimental conditions need to be finely adjusted to achieve better modeling effect.
The foregoing is directed to preferred embodiments of the present application, other than the limiting examples of the present application, and variations of the present application may be made by those skilled in the art using the foregoing teachings. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present application still belong to the protection scope of the technical solution of the present application.
Claims (6)
1. A method for establishing a bee Parkinson model is characterized by comprising the following steps:
animal selection: selecting mature bees as an object for establishing a bee Parkinson model;
preparing a rotenone sucrose pollen solution: dissolving rotenone in dimethyl sulfoxide, and mixing with sucrose pollen solution to obtain rotenone sucrose pollen solution;
establishing a model: and feeding the mature bee with the rotenone sucrose pollen solution for more than 3 days to obtain the bee Parkinson model.
2. The method for building a bee parkinson's model according to claim 1, wherein: the concentration of the rotenone in the rotenone sucrose pollen solution is 50 mu mol/L-250 mu mol/L, and the sucrose pollen solution is prepared by putting pollen into a sucrose solution with the mass volume concentration of 40-60% according to the proportion that the adding amount of the pollen is 25-50% of the volume of the sucrose solution.
3. The method for establishing the bee parkinson model according to claim 1, wherein: the mature bees are 2 to 15 days old, and the bees are bees or bumblebees.
4. The use of the bee parkinson model constructed by the method for establishing the bee parkinson model as defined in any one of claims 1 to 3 for screening drugs for preventing or/and treating parkinson's disease.
5. Use of the bee parkinson's model constructed by the method for constructing a bee parkinson's model according to any one of claims 1 to 3 for studying the mechanism of parkinson's associated diseases.
6. Use according to claim 4, characterized in that: kenyang cells from the bee Parkinson model were used for drug screening.
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