Method for constructing parkinson's disease-like animal models

Abstract

The application discloses a kind of Parkinson's disease sample animal model construction method, comprising the following steps: 1) determine the function gene of cholesterol sulfation modification, according to the sequence and transcript site of the gene of the animal determines interference target point;2) for the tissue to be interfered, prepare the injection adeno-associated virus of the animal of specific serotype;3) using the adeno-associated virus of step 2) intervention the animal, obtain Parkinson's disease sample animal model.The application provides a kind of new Parkinson's disease sample experimental animal model high-efficiency simple preparation method, can be used for Parkinson's disease animal model construction, pathogenesis analysis, food and drug screening, precision nutrition and old age health management etc. Research scene.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of animal pathology, and particularly relates to a Parkinson's disease-like animal model construction method and application thereof. BACKGROUND

[0002] With the extension of the average life expectancy, the incidence and number of people suffering from age-dependent, progressive neurodegenerative diseases are increasing year by year. Parkinson's disease (PD), also known as paralysis agitans, is the second largest neurodegenerative disease in the world, with the fastest growth in number and mortality among neurological diseases, and is also the main cause of disability and disability in the elderly population, seriously affecting the quality of life of the elderly and bringing huge social welfare pressure and economic burden of care.

[0003] Parkinson's disease is a neurodegenerative disease characterized by the degeneration of dopaminergic neurons and abnormal aggregation of alpha-synuclein, with a complex and highly heterogeneous pathological mechanism. The typical symptoms are progressive movement disorders, including tremor, bradykinesia, rigidity, postural instability and gait difficulty.

[0004] Rodents are easy to monitor under laboratory conditions and have wide application value in the biomedical field. The existing Parkinson's disease pathological models derived from neurotoxins, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 6-hydroxydopamine, rotenone, paraquat, etc., can quickly cause movement disorder phenotypes, but have the disadvantages of pathological deficiency and drug resistance, resulting in unstable effects, easy death and other problems.

[0005] The existing genetic models can simulate the pathological mechanism of alpha-synuclein, but it is difficult to form a behavioral phenotype. The existing Parkinson's disease-like genetic animal model construction process is complex, the movement disorder phenotype is unstable, and the pathological mechanism is not uniform. SUMMARY

[0006] To solve the above problems, the application provides a Parkinson's disease-like animal model construction method based on gene editing technology, comprising the following steps:

[0007] 1) Determine the function gene of cholesterol sulfation modification, and determine the interference target according to the sequence and transcript site of the gene of the animal;

[0008] 2) For the tissue to be interfered, prepare an injection adeno-associated virus of the animal of a specific serotype;

[0009] 3) Use the adeno-associated virus of step 2) to intervene in the animal to obtain a Parkinson's disease-like animal model.

[0010] The application first determines the target gene and the interference target.

[0011] Cholesterol is an important component of the brain, and the content in the brain is the highest, the content of cholesterol in the brain accounts for about 20% of the total amount of cholesterol in the body. As early as the 1960s, it was found that cholesterol sulfate was distributed in tissues such as the brain, but its biological function in the brain has not been elucidated. Cholesterol is not only an important structural component of cell membranes and myelin sheaths, but also a precursor substance of various metabolites such as cholesterol sulfate (Cholesterol Sulfate, CS). Cholesterol plays an important role in the formation of synapses and the transmission of nerve signals.

[0012] Cholesterol sulfate is catalytically synthesized by hydroxysteroid sulfotransferase family 2B member 1 (Hydroxysteroid Sulfotransferase Family 2B member 1, SULT2B1) with cholesterol as a substrate. Cholesterol sulfate, as a product generated by hydroxysteroid sulfotransferase family 2B member 1 catalysis of cholesterol, is mainly distributed in synapses and is crucial for brain neural activity.

[0013] In the present application, the gene responsible for the sulfation modification of cholesterol can be hydroxysteroid sulfotransferase family 2B member 1 gene sult2b1.

[0014] The transcript sequence of the functional gene responsible for the sulfation modification of cholesterol of the target animal is obtained from the National Center for Biotechnology Information (NCBI) Gene database, and the possible effective gene interference target is determined by means of the gene knockout, knockdown and expression-pre-designed shRNA webpage of Sigma Aldrich Trading Co., Ltd. (https: / / www.sigmaaldrich.cn / CN / zh / semi-configurators / shrna). In combination with further experiments, the effective interference target is determined.

[0015] Further, the purpose of the construction of the Parkinson's disease-like animal model of the present application is to target gene editing of brain tissue and inhibit lipid sulfation modification. Therefore, the target tissue to be subjected to gene interference is the brain.

[0016] Further, according to the tissue specificity of the packaging virus for the brain, a suitable packaging plasmid of the serotype is selected. Therefore, in the present application, the serotype of the packaging plasmid for the brain is determined to be PHP.eB.

[0017] Further, the animal injection adeno-associated virus containing the interference target is prepared by a cell culture method.

[0018] Furthermore, by injection, the prepared adeno-associated virus containing the interfering target was used to intervene in the animals to obtain Parkinson's disease-like model animals.

[0019] Furthermore, based on the pathological characteristics of Parkinson's disease, which is centered on dopamine disorder, and the possible effects of cholesterol sulfate on synaptic function and metabolism, the pathological mechanism explored in this invention mainly revolves around the metabolism and transport of neurotransmitters, especially dopamine.

[0020] The present invention also provides a Parkinson's disease-like animal model constructed by the above-described method for constructing a Parkinson's disease-like animal model.

[0021] This invention also provides applications of the above-mentioned Parkinson's disease-like animal models, including the analysis of the pathogenesis of Parkinson's disease, the screening of foods and drugs for Parkinson's disease, and the provision of precision nutrition and health management for Parkinson's disease.

[0022] Furthermore, this invention has discovered that cholesterol sulfate, a product catalyzed by cholesterol via hydroxysteroid sulfate transferase 2B1, can improve motor dysfunction in Parkinson's-like animal models.

[0023] The present invention also provides the use of cholesterol sulfate in improving Parkinson's-like movement disorders.

[0024] Furthermore, in the above-mentioned uses, the amount of cholesterol sulfate used is 100 mg / kg body weight.

[0025] Furthermore, the animal used in the animal model of the present invention can be a mouse.

[0026] Furthermore, through web design and experimental screening, the interference targets for mice were identified as sequences Seq_1, Seq_2, and / or Seq_3 in the sequence listing.

[0027] Furthermore, the prepared adeno-associated virus was used to intervene in mice via tail vein injection or in situ injection to obtain Parkinson's disease-like model mice.

[0028] Furthermore, the preferred intervention method is tail vein injection, which can effectively avoid damage to mice during the modeling process.

[0029] The present invention also provides a Parkinson's disease-like mouse model constructed using the above-described method for constructing a Parkinson's disease-like animal model.

[0030] Parkinson's disease-like mice constructed using the Parkinson's disease-like animal model of the present invention exhibited significant motor dysfunction phenotypes and dopamine-related core pathological mechanisms of Parkinson's disease after inhibiting cholesterol sulfation in the brain.

[0031] Furthermore, behavioral analyses of motor disorder phenotypes such as rotarod, crossbar, grid, and trajectory analysis were performed on the model mice to evaluate the degree of motor disorders such as tremor, bradykinesia, rigidity, postural instability, and gait difficulty.

[0032] Furthermore, gene expression analysis and other methods were used to explore the pathological mechanisms of movement disorders in the model mice.

[0033] Furthermore, using the commonly used 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease model mouse as an example, the ameliorative effect of cholesterol sulfate was explored.

[0034] Furthermore, MPTP-induced Parkinson's disease model mice were supplemented with cholesterol sulfate in their diet, and their motor performance was evaluated using rotarods, grids, and crossbars to explore the effect of dietary cholesterol sulfate supplementation on the motor performance of Parkinson's disease model mice.

[0035] Furthermore, to verify the role of intracranial cholesterol sulfation in Parkinson's disease, MPTP-induced Parkinson's disease-like mice were used, and dietary supplementation with cholesterol sulfate significantly improved the motor performance of MPTP-induced Parkinson's disease model mice.

[0036] Specifically, the present invention is as follows.

[0037] 1. A method for constructing a Parkinson's disease-like animal model, comprising the following steps:

[0038] 1) Identify the functional genes that modify cholesterol sulfation, and determine the interference targets based on the sequence and transcript sites of the genes in the animals;

[0039] 2) Prepare adeno-associated virus for injection of a specific serotype from the animal targeted for interference;

[0040] 3) Intervene the animals with the adeno-associated virus from step 2) to obtain a Parkinson's disease-like animal model.

[0041] 2. The method for constructing a Parkinson's disease-like animal model as described in item 1, characterized in that: the gene responsible for cholesterol sulfation modification in step 1) is the hydroxysteroid sulfate transferase 2B1 gene.

[0042] 3. The method for constructing a Parkinson's disease-like animal model as described in item 1, characterized in that: the target tissue for gene expression interference in step 2) is the brain.

[0043] 4. The method for constructing a Parkinson's disease-like animal model as described in item 3, characterized in that: the specific serotype of the brain-specific packaging plasmid in step 2) is PHP.eB.

[0044] 5. The method for constructing a Parkinson's disease-like animal model as described in any one of items 1 to 4, characterized in that: the animal is a mouse.

[0045] 6. The method for constructing a Parkinson's disease-like animal model as described in item 5, characterized in that: the interference target sequence of the gene in step 1) is the sequence Seq_1, Seq_2 and / or Seq_3 in the sequence listing.

[0046] 7. A Parkinson's disease-like animal model constructed using the construction method described in any one of items 1 to 6.

[0047] 8. The uses of the Parkinson's disease-like animal model described in item 7 include: elucidating the pathogenesis of Parkinson's disease, screening foods and drugs for Parkinson's disease, and providing precision nutrition and health management for Parkinson's disease.

[0048] 9. The use of cholesterol sulfate, a product of cholesterol catalysis by hydroxysteroid sulfate transferase 2B1, in improving Parkinson's-like movement disorders.

[0049] 10. The use as described in item 9, characterized in that: the amount of cholesterol sulfate used is 100 mg / kg body weight.

[0050] The Parkinson's disease-like animal model constructed using the method of this invention can inhibit cholesterol sulfation in the brain and possesses the core dopamine-related pathological mechanisms. The Parkinson's disease-like animal model constructed based on gene editing technology provided by this invention has the advantages of stable motor disorder phenotypes and short construction cycle, and can be widely applied in research scenarios such as the construction of Parkinson's disease-like animal models, pathogenesis analysis, food and drug screening, precision nutrition, and aging health management.

[0051] The beneficial effects of this invention are as follows:

[0052] (1) This invention provides a new, efficient and simple method for preparing a Parkinson's disease-like experimental animal model, which can be used in research scenarios such as the construction of Parkinson's disease-like animal models, the analysis of pathogenesis, food and drug screening, precision nutrition and healthy aging.

[0053] (2) This invention clarifies the core target of inhibiting cholesterol sulfation in the brain to construct an experimental animal model of Parkinson's disease, providing a new approach for healthy aging and precision nutrition.

[0054] (3) This invention provides a possible mechanistic explanation for the construction of a Parkinson's disease-like experimental animal model by inhibiting cholesterol sulfation in the brain, and provides a theoretical basis for research scenarios such as the analysis of the pathogenesis of Parkinson's disease and the screening of food and drugs.

[0055] (4) This invention has discovered the effect of dietary cholesterol sulfate supplementation on improving Parkinson’s disease-like motor disorders in animals, and provides a new approach to precise nutritional management of Parkinson’s disease. Attached Figure Description

[0056] Figure 1 This indicates the relative expression abundance of the sult2b1 gene in a gene target screening experiment according to an embodiment of the present invention.

[0057] Figure 2 The graph illustrates the changes in motor coordination and balance in mice according to an embodiment of the present invention.

[0058] Figure 3 The graph illustrates the change in mouse muscle strength according to an embodiment of the present invention.

[0059] Figure 4 The curve represents the change in the ability of a mouse to repair an externally forced posture according to an embodiment of the present invention.

[0060] Figure 5 This represents the test results of the mouse's motor ability according to an embodiment of the present invention.

[0061] Figure 6 This indicates the relative abundance of gene expression related to interference targets, dopamine production, and transport genes in one embodiment of the present invention.

[0062] Figure 7 This invention illustrates the effect of dietary cholesterol sulfate supplementation on the motor performance of MPTP Parkinson's disease model mice, according to one embodiment of the present invention. Detailed Implementation

[0063] To better understand this invention, the following embodiments are provided in conjunction with the accompanying drawings. It should be understood that the embodiments of this invention are for illustrative purposes only and not for limiting the invention; the scope of protection of this invention is defined solely by the claims. The embodiments provided are merely preferred embodiments and are not intended to limit the invention in any way. Those skilled in the art can make changes, equivalent substitutions, or modifications based on the content of this invention to form different implementations. However, any changes and modifications, and any equivalent substitutions made to the method of this invention without departing from the inventive concept are within the scope of protection of this invention.

[0064] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0065] The embodiments of the present invention are illustrated using experimental animals, such as mice.

[0066] Example 1: Identifying the target gene and interference target

[0067] The transcript sequence of the sult2b1 gene, responsible for cholesterol sulfation modification in the mouse genome, was obtained from the NCBI Gene database. Using the gene knockout, knockdown, and expression—pre-designed shRNA webpage (https: / / www.sigmaaldrich.cn / CN / zh / semi-configurators / shrna) from Sigma-Aldrich Trading Co., Ltd., three potentially effective sult2b1 gene interference target sequences were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd. for target screening. The three target sequences were named S1, S2, and S3, and the randomized control sequence was named Con.

[0068] The sequences of the three target sequences for interfering with sult2b1 gene expression, S1, S2, and S3, and the random control sequence Con, are as follows.

[0069] S1:5'-CCTAAGATTGCTGGGCAATTAA-3' Seq_1

[0070] S2:5'-AACAGTGTTTACCGAGAGCAA-3' Seq_2

[0071] S3:5'-AAGGTGAATACTTCAGATACAA-3' Seq_3

[0072] Con: 5'-CCTAAGGTTAAGTCGCCCTCG-3' Seq_4

[0073] Mouse hippocampal neuron cell line HT22 (purchased from Nanjing Kebai Biotechnology Co., Ltd., catalog number: CBP61631) was expanded in DMEM high-glucose medium containing 10% fetal bovine serum (DMEM high-glucose, containing GlutaPlus, containing sodium pyruvate, and without HEPES, purchased from Wuhan Saiwei Biotechnology Co., Ltd., catalog number: G4511-500ML). The culture was carried out at 1.5 × 10⁻⁶ cells / mL. 5Cells were seeded into 12-well cell culture plates at a concentration of cells / mL. The next day, when the cell confluence reached 70-80%, the Lipofectamine™ 3000 transfection reagent (purchased from Thermo Fisher Scientific (China) Co., Ltd., catalog number: L3000001) was used to transfect three target sequences for interfering with sult2b1 gene expression (S1, S2, S3) and a random control sequence (Con) into serum-free DMEM high-glucose culture medium. Each sequence was divided into three replicates. After incubation for 4 h, the medium was replaced with DMEM high-glucose culture medium containing 10% fetal bovine serum and cultured for another 48 h.

[0074] RNA was extracted from cells using the Trizol method. 400 μL of Trizol lysis buffer was added to each cell culture well and incubated at room temperature for 5 min. Cells were then continuously pipetted until complete lysis. The cell lysis buffer was transferred to a 1.5 mL centrifuge tube, and 0.2 volumes of chloroform were added according to the TRIzol reagent (Thermo Fisher Scientific (China) Co., Ltd., catalog number: 15596018CN). After centrifugation, the supernatant was collected, and an equal volume of isopropanol was added. The mixture was vortexed and centrifuged again. The precipitate was washed with pre-chilled 75% ethanol at -20°C. After centrifugation, the supernatant was discarded, and the precipitate was dissolved in DEPC water (DNase, RNase free) (purchased from Shanghai Beyotime Biotechnology Co., Ltd., product number: R0021) to obtain the cellular RNA.

[0075] Take 2 μg of cellular RNA and synthesize it according to the cDNA-strand synthesis kit (OneScript). ® Plus Reverse Transcriptase (purchased from Zhenjiang Aibimeng Biotechnology Co., Ltd., product code: G237) was used for reverse transcription to obtain cDNA. qPCR amplification was performed according to the instructions of BlasTaq™ 2X qPCR MasterMix (purchased from Zhenjiang Aibimeng Biotechnology Co., Ltd., product code: G892). The PCR reaction program was: 95℃ pre-denaturation for 10 min, followed by 45 cycles of 95℃ for 15 s, 55℃ for 20 s, and 72℃ for 30 s. β-actin was used as an internal control to correct for the mRNA expression level of the target gene.

[0076] The primer sequences for sult2b1 and β-actin for real-time quantitative detection of gene expression are as follows.

[0077] sult2b1-F:5'-GCGAGACCATCATAAGCG-3' Seq_5

[0078] sult2b1-R:5'-GTAAATCACCTTAGCTTGGA-3' Seq_6

[0079] β-actin-F: 5'-CAGGCATTGCTGACAGGATG-3' Seq_7

[0080] β-actin-R: 5'-TGCTGATCCACATCTGCTGG-3' Seq_8

[0081] The relative expression abundance of the sult2b1 gene in cells is as follows: Figure 1 As shown. Figure 1 In this study, experimental results are expressed as Mean ± SEM. One-way ANOVA-LSD test was used for significance analysis. A p-value < 0.05 was considered statistically significant between groups. Different letters above the data bars indicate statistically significant differences between data points.

[0082] like Figure 1 As shown, compared with the control Con sequence, the S1, S2, and S3 target sequences all significantly reduced the expression of the sult2b1 gene in cells. The S2 target sequence significantly reduced the expression of the sult2b1 gene in cells by 73.0%, showing the best effect.

[0083] Example 2: Preparation of Interference Virus

[0084] The following section uses the S2 sequence as an example to conduct subsequent gene interference experiments.

[0085] Identifying the target tissue for gene interference. In this invention, the target tissue for gene expression interference is the brain.

[0086] Based on the tissue specificity of the packaging virus, a suitable serotype of packaging plasmid is selected. In this invention, the serotype of the packaging plasmid targeting the brain is PHP.eB.

[0087] Adeno-associated virus for injection of a specific serotype in mice was prepared using cell culture methods.

[0088] The target sequence was the adeno-associated virus construct plasmid (dsAAV.CAG.EGFP-miR30-shRNA1(sult2b1).WPRE.SV40pA) that interfered with the expression of the sult2b1 gene, as shown in Seq_2 in the sequence listing. The control plasmid (dsAAV.CAG.EGFP.WPRE.SV40pA) without the target gene was purchased from Guangzhou Paizhen Biotechnology Co., Ltd. The packaging plasmids pHelper (catalog number: P0243) and pUCmini-iCAP-PHP.eB (catalog number: P12266) were purchased from Wuhan Miaoling Biotechnology Co., Ltd.

[0089] The plasmid purification method is as follows. Take 1 μL of each of the four plasmids (50 ng / μL) and incubate them together with DH5α supercompetent cells (purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number: D1031S) on ice for 15–30 min. Then, heat shock them in a 42℃ water bath for 90 s, and immediately transfer them to ice for 2–3 min to obtain the transformation solution. Take 70–80 μL of the transformation solution and spread it evenly on LB broth containing 0.01% ampicillin using a spreader. Invert the plates and incubate overnight at 37℃. The next day, pick single colonies and incubate them in LB broth containing 0.01% ampicillin at 37℃ with constant temperature shaking at 220 rpm for approximately 12 h. Extract plasmids from the obtained bacterial culture according to the instructions of the plasmid miniprep kit (purchased from Tiangen Biotech (Beijing) Co., Ltd., catalog number: DP103).

[0090] The packaging method for adeno-associated virus (AAV) is as follows. Healthy human embryonic kidney cells (HEK239T, purchased from Nanjing Kebai Biotechnology Co., Ltd., model: CBP60439) were selected for AAV virus packaging. After expansion using DMEM high-glucose medium containing 10% fetal bovine serum, the cells were packaged at a rate of 1×10⁻⁶. 5 HEK239T cells were seeded into T25 culture flasks at a concentration of cells / mL. The next day, when the cell confluence reached 70-80%, the control plasmid dsAAV.CAG.EGFP.WPRE.SV40pA was mixed with the packaging plasmids pHelper and pUCmini-iCAP-PHP.eB at a ratio of 1:1:1 according to the instructions of polyethyleneimine (linear PEI transfection reagent (fast-dissolving type) MW40000, purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number: 40816ES01). The target plasmid dsAAV.CAG.EGFP-miR30-shRNA1(sult2b1).WPRE.SV40pA was also mixed with the packaging plasmids pHelper and pUCmini-iCAP-PHP.eB. The two plasmid systems were then added to serum-free DMEM high-glucose culture medium, gently mixed, and incubated for 6-8 days. After h, the medium was replaced with DMEM high-glucose medium containing 10% fetal bovine serum, and cultured for another 72 h.

[0091] The purification method for adeno-associated virus (AAV) is as follows. Cell supernatant was collected and filtered through a 0.45 μm filter to remove cell debris. Iodixanol gradient medium (OptiPrep™ Density Gradient Medium, purchased from Sigma-Aldrich, product number: D1556) was added, and the mixture was centrifuged at 60,000 g for 3 h at 4°C. The viral layer was collected and resuspended in physiological saline to obtain control virus without the sult2b1 interfering sequence and sult2b1 interfering virus, respectively. The titer was determined and adjusted according to the instructions of the Takara Lenti-X™ GoStix™ Lentiviral Titer Assay Kit (purchased from Baori Biotechnology (Beijing) Co., Ltd., catalog number: 631280), with a final titer of 1×10⁻⁶. 12 The titer of VG / mL is used for animal experiments.

[0092] Example 3: Construction of a Parkinson's Disease-like Mouse Model

[0093] Mice were treated with the prepared adeno-associated virus via tail vein injection to obtain Parkinson's disease-like mice.

[0094] Male C57BL / 6J mice (8 weeks old) were housed indoors at 20±2℃, 60% relative humidity, and a 12h / 12h light / dark cycle, with free access to stick-shaped maintenance feed and water. After one week of acclimatization, they were randomly divided into two groups of 10 mice each, based on their body weight, and fed 3×10⁻⁶ feeds. 11 The dose of VG / animal was administered via tail vein injection of control virus without sult2b1 interference sequence and sult2b1 interference virus, respectively.

[0095] Using a brain-targeting capsid protein carried by the virus, the plasmid crosses the blood-brain barrier and enters brain cells. The hairpin structure of the inserted fragment is recognized by intracellular endonucleases and cleaved into small interfering RNA (SRNA). This SRNA then binds to endonucleases, exonucleases, and helicases to form an RNA-induced silencing complex, which specifically binds to and cleaves the sult2b1 gene mRNA, thereby blocking sult2b1 gene expression. Once viral transfection takes effect, i.e., sult2b1 gene expression stabilizes at a low level, the Parkinson's disease-like mouse model is successfully established.

[0096] Example 4: Behavioral Evaluation

[0097] 4.1 Rotating Rod Experiment

[0098] Under constant temperature and dark conditions, the mouse trunk and limbs were placed stably on the surface of a rotating bar (initially at 4 rpm, linearly increased to 20 rpm / min, then timing was started). The time of the mouse's first fall, i.e., the balance time, was recorded. Each mouse was measured three times, with each test 15 min apart, and the average value was taken. The longest recorded time was 300 s. By comparing the balance times between groups, the changes in the mice's motor coordination and balance ability were analyzed.

[0099] The motor coordination and balance ability of mice were evaluated using the rotarod test, and the balance time change curve is shown in Figure 1. Figure 2 As shown. Figure 2 In this study, experimental results are expressed as Mean±SEM. Student's t-test was used for significance analysis. Results were considered statistically significant with P<0.05, and were marked to the right of the data points. "*" indicates P<0.05 (significant difference), "**" indicates P<0.01 (highly significant difference), and "***" indicates P<0.001 (extremely significant difference).

[0100] Starting on day 8 of modeling, the balance time of the model group showed a downward trend. On day 9, the balance time of the model group (mice with sult2b1 gene interference) was significantly reduced by 11.4% compared to the control group mice, which had the same experimental conditions except for sult2b1 gene interference. Starting on day 10, highly significant differences in motor coordination and balance ability appeared between the two groups of mice, and the differences gradually widened. By day 14, the balance time of the model group had decreased to 15.8% of that of the control group.

[0101] 4.2 Grid Experiment

[0102] The experimental setup consisted of a 12 cm × 12 cm horizontal grid woven from stainless steel wire, with mesh openings of 0.5 cm × 0.5 cm. Mice were placed in the center of the grid. Once the mice gripped the grid with their paws, the grid was slowly rotated 180 degrees, suspending the mice upside down. Timing was started and stopped when the mice fell, serving as the drop delay time. Each mouse underwent the test three times, with 15-minute intervals between each test. The average drop delay time was taken, and the longest possible drop delay time was set at 120 seconds. Changes in muscle strength in the mice were analyzed by comparing the drop delay times between groups.

[0103] The muscle strength of the mice was evaluated using a grid test, and the change curve of the drop delay time is shown in the figure below. Figure 3 As shown. Figure 3In this study, experimental results are expressed as Mean±SEM. Student's t-test was used for significance analysis. Results were considered statistically significant with P<0.05, and were marked to the right of the data points. "*" indicates P<0.05 (significant difference), "**" indicates P<0.01 (highly significant difference), and "***" indicates P<0.001 (extremely significant difference).

[0104] Starting on day 8 of modeling, the drop delay time in the model group showed a downward trend with significant differences. On day 8, the drop delay time in the model group was significantly reduced by 19.6% compared to the control group. Starting on day 9, a highly significant difference in muscle strength was observed between the two groups of mice, and the difference gradually widened. By day 14, the drop delay time in the model group was reduced by 85.7% compared to the control group.

[0105] 4.3 Stiff crossbar test

[0106] Before the test, mice were placed in the experimental environment for 30 minutes to acclimatize and reduce stress. The height of the horizontal bar was fixed at 5 cm. During the test, the mouse's two front paws were gently placed in the center of the horizontal bar, keeping the mouse in a semi-upright posture, and the timer was started. The timer was stopped when the mouse removed one front paw from the bar, and this was recorded as the rigidity time. Each mouse was tested three times, and the average value was taken. If the mouse did not move its limbs within 180 seconds, the maximum rigidity time was recorded as 180 seconds. By comparing the rigidity times between groups, the mice's ability to recover from externally forced postures was analyzed.

[0107] The ability of mice to repair externally forced postures was evaluated by the rigidity bar test, and the rigidity time change curve is shown in Figure 1. Figure 4 As shown. Figure 4 In this study, experimental results are expressed as Mean±SEM. Student's t-test was used for significance analysis. Results were considered statistically significant with P<0.05 and marked to the right of the data points. "*" indicates P<0.05 (significant difference), "**" indicates P<0.01 (highly significant difference), and "***" indicates P<0.001 (extremely significant difference).

[0108] Starting on day 5 of modeling, the rigidity time of the model group mice began to increase, fluctuated and decreased on day 10, and then rapidly increased until the end of modeling. On day 7, the rigidity time of the model group was 1.86 times that of the control group; on day 9, it increased to 4.61 times that of the control group; on day 10, it improved and was 3.40 times that of the control group; subsequently, the severity of rigidity increased rapidly, gradually increasing from 3.41 times that of the control group on day 11 to 11.0 times that of the control group on day 14.

[0109] 4.4 Motion Trajectory Analysis:

[0110] On day 11 of modeling, the mice were gently placed on a smooth, flat table, and their movement trajectory was recorded by video. The video images were exported frame by frame at 0.1 s intervals, and the changes in the mice's motor ability were analyzed by comparing the distance the mice traveled (measured from the base of their tails) and their gait within 1 second.

[0111] The mice's motor abilities were demonstrated by video recordings of their behavioral trajectories, with frame-by-frame images at 0.1-second intervals, as shown below. Figure 5 As shown in the image, within the same 1 second, the control group mice moved a distance of approximately 2 body lengths, while the model group mice moved only about 0.5 body lengths. Furthermore, analysis of the movement shown in the images revealed that the control group mice exhibited a stable alternating forward and backward movement of their hind limbs, while the model group mice had their hind limbs positioned on either side of their bodies, indicating insufficient alternation and unstable support.

[0112] Example 5: RT-PCR gene expression analysis

[0113] After the behavioral tests were completed, the mice were killed and their brains were collected on day 15. After decapitation, the brains of the mice were quickly dissected on ice, cut in half along the midline, wrapped in aluminum foil and immersed in liquid nitrogen, and then stored in an ultra-low temperature freezer at -80°C.

[0114] RNA was extracted from the right hemisphere of the mouse brain using the Trizol method. Brain tissue was removed from a -80°C cryogenic freezer, weighed, and 10 times its volume of lysis buffer (Invitrogen TRIzol reagent, purchased from Thermo Fisher Scientific (China) Co., Ltd., catalog number: 15596018CN) was added. 3 mm zirconium oxide beads were added to a cryogenic tissue homogenizer, and homogenization was performed at 4°C and 60 Hz for 30 s, followed by standing at room temperature for 5 min. After centrifugation, the uppermost lipid layer was removed, and the supernatant was collected. Chloroform was added to the supernatant, and the mixture was vortexed and centrifuged again, collecting the supernatant. Isopropanol was added, and the mixture was vortexed again and centrifuged once more, collecting the precipitate. The precipitate was resuspended in pre-cooled 75% ethanol at -20°C, centrifuged, and the supernatant was collected. The precipitate was dissolved in DEPC water (DNase, RNase free) (purchased from Shanghai Beyotime Biotechnology Co., Ltd., product number: R0021) to obtain RNA.

[0115] The gene expression of hydroxysteroid sulfate transferase 2B1 (SULT2B1), tyrosine hydroxylase (TH), and dopamine transporter (DAT) in mouse brain tissue was measured, with β-actin as an internal reference gene.

[0116] All primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the primer sequences are as follows.

[0117] sult2b1-F:5'-GCGAGACCATCATAAGCG-3' Seq_5

[0118] sult2b1-R:5'-GTAAATCACCTTAGCTTGGA-3' Seq_6

[0119] β-actin-F: 5'-CAGGCATTGCTGACAGGATG-3' Seq_7

[0120] β-actin-R: 5'-TGCTGATCCACATCTGCTGG-3' Seq_8

[0121] th-F: 5'-CCACGGTGTACTGGTTCACT-3' Seq_9

[0122] th-R: 5'-GGCATAGTTCCTGAGCTTGT-3' Seq_10

[0123] dat-F:5'-GAGGTTTCCCTACCTGTGCT-3' Seq_11

[0124] dat-R:5'-GTGAAGCCCACACTTTCAG-3' Seq_12

[0125] Take 2 μg of mouse brain tissue RNA and synthesize it according to the cDNA-strand synthesis kit (OneScript). ® Plus Reverse Transcriptase (purchased from Zhenjiang Aibimeng Biotechnology Co., Ltd., product code: G237) was used for reverse transcription to obtain cDNA. qPCR experiments were performed according to the instructions of the BlasTaq 2X qPCR MasterMix (purchased from Zhenjiang Aibimeng Biotechnology Co., Ltd., product code: G892). The PCR reaction program was: 95℃ pre-denaturation for 10 min, with one cycle consisting of 95℃ for 15 s, 55℃ for 20 s, and 72℃ for 30 s, repeated for 45 cycles. β-actin was used as an internal control to correct for the mRNA expression level of the target gene.

[0126] Gene expression of hydroxysteroid sulfate transferase 2B1 (SULT2B1), tyrosine hydroxylase (TH), and dopamine transporter (DAT) in mouse brain tissue is as follows: Figure 6 As shown. Figure 6In this study, experimental results are expressed as Mean ± SEM. Student's t-test was used for significance analysis, and results with P < 0.001 were considered highly significant between groups, indicated by "***" above the data bars.

[0127] Sulfate transferases are responsible for catalyzing the transfer of sulfate groups from the sulfate donor adenosine 3'-phosphate-5'-phosphate sulfate (PAPS) to various receptor molecules in vivo. Among them, hydroxysteroid sulfate transferase 2B1 (SULT2B1) is responsible for the sulfation of cholesterol 3-hydroxyl groups. Figure 6 The results showed that the expression level of the hydroxysteroid sulfate transferase 2B1 gene sult2b1 in the model group decreased significantly by 54.1%. Tyrosine hydroxylase (TH) is involved in the conversion of tyrosine to dopamine. Figure 6 The results showed that the expression of the tyrosine hydroxylase gene th was significantly reduced by 45.2% in the model group. The dopamine transporter (DAT) is primarily responsible for re-uptake of dopamine from the synaptic cleft back to the presynaptic nerve ending, thereby regulating dopamine availability. Figure 6 The test results showed that the expression of the dopamine transporter gene dat decreased significantly by 70.5% in the model group.

[0128] In summary, behavioral analyses of rotarods, grids, crossbars, and movement trajectories revealed that mice lacking cholesterol sulfation modification in their brains exhibited significant progressive motor dysfunction, including tremor, bradykinesia, rigidity, postural instability, and gait difficulty, with symptom severity increasing with prolonged modeling time. Furthermore, expression analysis of key genes showed significant inhibition of dopamine synthesis and reuptake, consistent with Parkinson's disease pathology.

[0129] Example 6: Effects of dietary cholesterol sulfate supplementation on motor performance in MPTP Parkinson's disease model mice

[0130] Thirty male C57BL / 6J mice (8 weeks old) were housed indoors at 20±2℃, 60% relative humidity, and a 12h / 12h light / dark cycle, with free access to stick-shaped maintenance feed and water. After one week of acclimatization, they were randomly divided into three groups according to their body weight: a model group (MPTP) receiving intraperitoneal injection of MPTP at a dose of 30 mg / kg body weight for 5 consecutive days; a test substance group (CS) receiving intraperitoneal injection of MPTP at a dose of 30 mg / kg body weight for 5 consecutive days followed by gavage administration of cholesterol sulfate at a dose of 100 mg / kg body weight; and a normal control group (Nor) receiving both intraperitoneal injection and gavage administration of an equal volume of physiological saline. Motor function was assessed on day 6.

[0131] The effect of dietary cholesterol sulfate supplementation on motor performance in MPTP Parkinson's disease model mice, such as Figure 7 As shown. Figure 7 A, Figure 7 B. Figure 7 C represents the equilibration time of the swivel bar experiment, the fall delay time of the grid experiment, and the stiffness time of the rigid bar experiment, respectively. Figure 7 In this study, experimental results are expressed as Mean ± SEM. Student's t-test was used for significance analysis, and a p-value < 0.05 was considered statistically significant. Different letters above the data bars represent significant differences between groups.

[0132] Continuous MPTP injection is a common method for establishing a mouse model of Parkinson's disease, inducing significant motor impairment. Dietary supplementation with cholesterol sulfate (CS) significantly improved the motor performance of MPTP-treated Parkinson's model mice. Figure 7 As shown, MPTP significantly reduced the balance time of the rotarod test and the drop delay time of the grid test in mice by 70.5% and 55.8%, respectively, and significantly increased the stiffness time of the rigid bar test by 2.53 times. Dietary supplementation with cholesterol sulfate significantly increased the balance time of the rotarod test and the drop delay time of the grid test by 1.07 times and 85.0%, respectively, and significantly reduced the stiffness time of the bar test by 51.5%, respectively, on top of the MPTP model.

[0133] In summary, this invention, through targeted gene editing to inhibit the sulfation modification of cholesterol in the brain, can induce a stable motor disorder phenotype one week after targeted intervention, and this phenotype possesses the core pathological mechanism of dopamine deficiency. Furthermore, dietary supplementation with cholesterol sulfate can significantly improve the motor performance of an MPTP-induced Parkinson's mouse model.

Claims

1. A method for constructing a Parkinson's disease-like animal model, comprising the following steps: 1) The functional gene for cholesterol sulfation modification was identified as the hydroxysteroid sulfate transferase 2B1 gene. The interference target was determined based on the sequence and transcript site of the gene in mice. The interference target sequence of the gene is the sequence Seq_1, Seq_2 and / or Seq_3 in the sequence listing. The interference target sequence significantly reduced the expression of the hydroxysteroid sulfate transferase 2B1 gene. 2) Adeno-associated virus for injection in mice with a specific serotype was prepared for the purpose of interfering with the brain. The specific serotype of the packaging plasmid for the brain is PHP.eB. 3) The adeno-associated virus from step 2) was used to intervene in mice to obtain a Parkinson's disease-like mouse model.

2. Uses of the Parkinson's disease-like animal model constructed using the construction method of claim 1, including: Elucidation of the pathogenesis of Parkinson's disease, and screening of foods and drugs for Parkinson's disease.

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