Tree shrew Parkinson model construction method

Abstract

The invention discloses a tree shrew Parkinson model construction method which comprises the following steps: (1) weighing ceramide, and dissolving the ceramide in sterilized ultrapure water to prepare a solution with the concentration of 0.6 mg / mL; and (2) carrying out caudal vein injection on the tree shrew according to 4 mu g / g once per day for 7 days continuously, and normally feeding for 2 weeks after drug administration is finished to finish modeling. The stable tree shrew Parkinson's disease model can be established through caudal vein injection of ceramide with the dosage of 4 [mu] g / g for one week and normal feeding for two weeks after drug administration.

Description

Technical Field

[0001] The present invention belongs to the technical field of constructing disease animal models, and particularly relates to a method for constructing a tree shrew Parkinson model. Background Art

[0002] Parkinson's disease (PD), also known as paralysis agitans, is a clinical syndrome caused by multiple factors. It is the second most common neurodegenerative disease globally and one of the main causes of neurological dysfunction. The classic pathological manifestation of PD is the loss and even death of dopamine (DA)-ergic neurons in the substantia nigra compacta of the brain, resulting in a significant reduction in the content of striatal DA, and the abnormal aggregation of α-synuclein (α-syn) to form Lewy bodies, which causes the disease. The clinical manifestations are divided into motor symptoms: bradykinesia, stiffness, resting tremor, postural balance disorder, and non-motor symptoms: hyposmia, constipation, sleep disorders, depression, etc.

[0003] Research shows that the pathogenesis of PD is complex, and the etiology and pathogenesis are still unclear. However, a large amount of evidence indicates that oxidative stress, neuroinflammation, apoptosis, mitochondrial dysfunction, and proteasome dysfunction play important roles in the pathogenesis of PD.

[0004] PD animal models are powerful tools for elucidating the pathogenic mechanism of PD and exploring new pharmacological intervention methods. The current PD animal models are divided into two categories: neurotoxin-induced and genetic engineering. By introducing neurotoxins such as 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), paraquat, and rotenone, etc., PD models based on neurotoxins can be established and have been widely used. However, the existing animal models of Parkinson's disease have a long modeling time, and mainly use rodents (rats and mice) for modeling. Therefore, it is particularly important to develop an animal model with a closer genetic relationship to humans and a shorter modeling time. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for constructing a tree shrew Parkinson model. Tree shrews are highly similar to primates and even humans in terms of physiological anatomy, neural development, and psychological stress patterns, and are widely used in the field of biomedical research.

[0006] The purpose of the present invention is achieved as follows: (1) Weigh ceramide, dissolve it in sterilized ultrapure water, and prepare a solution with a concentration of 0.6 mg / mL. (2)Administer the solution described in step (1) to the tree shrews by tail vein injection at a dose of 4 μg / g, once a day for 7 consecutive days. After the administration, raise them normally for 2 weeks to complete the model establishment.

[0007] The beneficial effects of the present invention are as follows: In this application, a stable Parkinson's model of tree shrews can be established by tail vein injection of ceramide at a dose of 4 μg / g for 1 week and then raising them normally for 2 weeks after the administration. Description of the Drawings

[0008] Figure 1 It shows the changes in the expression of TH protein in 4 groups of tree shrews during the experiment of this application; Figure 2 It shows the changes in the expression of p-α-synuclein protein in 4 groups of tree shrews during the experiment of this application; Figure 3 It shows the TH immunofluorescence results of 4 groups of tree shrews during the experiment of this application; Figure 4 It shows the changes in the average fluorescence intensity of TH immunofluorescence in 4 groups of tree shrews during the experiment of this application. Detailed Embodiments

[0009] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments fall within the scope of protection of the present invention.

[0010] 1. Experimental Animals 15 ordinary-grade male tree shrews of Tupaia belangeri chinensis, with a body weight of 120 - 150 g, were purchased from the Institute of Medical Biology, Chinese Academy of Medical Sciences [SCXK (Yunnan) K2023 - 0003], and raised in the ordinary-grade animal house of Yunnan Luoyu Biotechnology Co., Ltd. [SYXK (Yunnan) K2021 - 0003]. They were raised in stainless steel cages at a room temperature of (24 ± 2) °C, a relative humidity of (50 ± 5) %, and a light / dark cycle of 12 h / 12 h.

[0011] 2. Main Reagents and Consumables Reagent Name Company Ceramide Sigma Tyrosine Hydroxylase (TH) Abcam p-α-synuclein Antibody Abcam Anti-rabbit IgG CST Goat Anti-Rabbit IgG H&L (DyLight® 488) Abcam RIPA Lysis Buffer (Strong) Beyotime Prestained Protein Marker Thermo Fisher Bovine Serum Albumin (BSA) Sigma SurperECLPlus Ultra-sensitive Luminescence PrimeSurface Fluorescent Mounting Medium (containing DAPI) ZSGB-BIO ZLI-9557 Blocking Goat Serum ZSGB-BIO ZLI-9021 PVDF Membrane Millipore 3. Main Instruments Instrument Name Company Nanophotometer Ultra-micro Spectrophotometer IMPLEN Protein Electrophoresis Cell, Electrotransfer Cell, Power Supply Bio-Rad ECL Luminescence Imager Tanon Fluorescence Microscope OLYMPUS BX53 4. Preparation of Reagents Preparation of ceramide: Weigh the required ceramide and dissolve it in sterilized ultrapure water to prepare a solution with a concentration of 0.6 mg / mL.

[0012] 5. Experimental Methods 5.1 Animal Model Making Experimental grouping: ① Normal group, ② Ceramide 2 μg / g group, ③ Ceramide 4 μg / g group, ④ Ceramide 8 μg / g group; In groups ② - ④, corresponding doses of ceramide solution were given by tail vein injection according to the body weight of the tree shrews according to the experimental grouping, once a day for 7 consecutive days; Group ① was given an equal volume of sterilized ultrapure water by tail vein injection.

[0013] 5.2 Behavioral observation of tree shrews After each injection, observe whether the tree shrews in each group show short-term symptoms (2 - 3 h) such as involuntary tremors, slow movement, unstable posture, limb stiffness, hair and tail erection, etc. Whether there are persistent symptoms such as overall decreased initiative (including feeding ability and activities, etc.) and slow movement, unsteady gait, clumsy movements, hunched back, and incoordination of forepaw movements.

[0014] 5.3 Detection After the tree shrews were given the drug for 7 days, they were normally fed for 2 weeks, and then the tree shrews were sacrificed for tissue sampling. After the tree shrews were anesthetized, the chest was opened and the heart was perfused with physiological saline. After observing that the liver turned white, the brain tissue of the tree shrews was taken out. The substantia nigra was isolated on ice and gently rinsed with 0.1 mol / L PBS. According to 500 μL of RIPA lysis buffer (containing 50 μL of protease inhibitor) added to every 100 mg of fresh tissue, after the tissue was fully minced with a sterile ophthalmic scissors, under an ice bath environment, it was homogenized with an ultrasonic cell disruptor for 20 s and left to lyse on ice for 20 min. Centrifuge at 12,000 rpm for 10 min at 4 °C, collect the supernatant, and pipette approximately 360 μL of the supernatant into each tube. Add 90 μL of 5×loading buffer (1 / 4 of the supernatant volume), mix well, centrifuge instantaneously for 10 s, and then boil in a 100 °C metal bath for 10 min to fully denature the protein. After the sample cooled down, the protein concentration was measured. Then SDS-PAGE protein electrophoresis was carried out. Electrophoresis was carried out at a constant voltage of 80 V until the lower edge of the sample contacted the separating gel (about 30 min), and then continued electrophoresis at a constant voltage of 130 V for nearly 2 h until the bromophenol blue just overflowed from the lower edge of the separating gel and then stopped electrophoresis; Wet transfer was carried out at 280 mA / 1 h for membrane transfer. Add 5% skim milk and block at room temperature for 2 h. Add the primary antibodies (rabbit anti-p-α-synuclein antibody 1:1000, rabbit anti-Tyrosine Hydroxylase antibody 1:5000) and mix overnight in a silent mixer in a 4 °C refrigerator. Wash the membrane 3 times with TBST buffer. Incubate with a horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:1000) at room temperature for 2 h. Wash the membrane 3 times with TBST buffer. Chemiluminescence method was used to add ECL reagent and develop in a gel imaging system.

[0015] 5.4 Immunofluorescence detection After the tree shrews were administered drugs for 7 days, they were normally fed for 2 weeks, and then the tree shrews were sacrificed for tissue sampling. After anesthesia, the tree shrews were perfused successively with normal saline and 4% paraformaldehyde solution through the heart. After the brain was removed, it was placed in 4% paraformaldehyde to prepare paraffin sections for subsequent immunofluorescence staining.

[0016] Immunofluorescence staining: The paraffin sections were deparaffinized to water. The sections were immersed in an antigen retrieval box containing citrate buffer (pH = 6.0) and antigen retrieval was performed in a microwave oven. After blocking with 5% goat serum for 1 h, the primary antibody (TH 1:500) was added and incubated overnight at 4 °C. Then, goat anti-rabbit IgG labeled with 488 (1:1000) was added and incubated for 2 h at room temperature in the dark. The sections were mounted with a mounting medium containing DAPI and observed and photographed under an upright fluorescence microscope.

[0017] 6. Experimental results 6.1 Behavioral manifestations of tree shrews induced by ceramide After 3 injections of 4 μg / g and 8 μg / g ceramide, the tree shrews began to show typical PD motor symptoms visible to the naked eye, including involuntary tremors, bradykinesia, postural instability, limb stiffness, hair and tail erection, etc. The visible abnormal manifestations generally appeared 1 - 3 min after ceramide injection and basically returned to normal within 2 h. With the increase in the number of injections, the PD motor symptoms became more obvious. After 6 injections of 2 μg / g ceramide, typical PD motor symptoms began to appear. There were no abnormal manifestations in the control group.

[0018] 6.2 Detection Compared with the normal group, the protein expression of p-α-synuclein in the ceramide group increased, and it was positively correlated with the dose, and the differences were all statistically significant (p < 0.05); compared with the normal group, the protein expression of Tyrosine Hydroxylase (TH) in the ceramide group decreased, and it was negatively correlated with the dose, but there was no significant difference between the 4 μg / g and 8 μg / g groups.

[0019] Table 1 Changes in the protein expressions of p-α-synuclein and TH in 4 groups of tree shrews during the experiment TH (Tyrosine Hydroxylase) p-α-synuclein NC 1.442±0.058 0.399±0.098 2μg / g Ceramide <![CDATA[1.202±0.056 b > <![CDATA[0.619±0.015 b > 4μg / g Ceramide <![CDATA[0.529±0.089 b,d > <![CDATA[1.116±0.077 b,d > 8μg / g Ceramide <![CDATA[0.458±0.109 b,d > <![CDATA[1.274±0.051 b,d,e > n = 3, , a P <0.05, b P <0.01, compared with the NC group; c P <0.05, d P <0.01, compared with the 2 μg / g Ceramide group; e P< 0.05, compared with the 4 μg / g Ceramide group. 6.3 Immunofluorescence results of TH Compared with the normal group, the average fluorescence intensity of immunofluorescence of Tyrosine Hydroxylase (TH) in the Ceramide group decreased, demonstrating that ceramide induced damage to dopamine neurons in tree shrews, and there was little difference between the 4 μg / g and 8 μg / g groups.

Claims

1. A method for constructing a tree shrew Parkinson's model, characterized in that: It includes the following steps: (1) Weigh ceramides and dissolve them in sterilized ultrapure water to prepare a solution with a concentration of 0.6 mg / mL; (2) Administer the solution described in step (1) to the tree shrews by tail vein injection at a dose of 4 μg / g once a day for 7 consecutive days. After the administration, raise them normally for 2 weeks to complete the model establishment.

2. The method for constructing a tree shrew Parkinson's model according to claim 1, characterized in that: The tree shrews mentioned are common-grade male Tupaia belangeri chinensis.

3. The method for constructing a tree shrew Parkinson's model according to claim 1, characterized in that: The body weight of the tree shrews is 120-150 g, and the breeding room temperature is 24±2°C, the relative humidity is 50±5%, and the light / dark cycle is 12 h / 12 h.

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