Construction method of tree shrew Alzheimer's disease model

Abstract

The invention discloses a method for constructing a tree shrew Alzheimer's disease model, which comprises the following specific steps of: (1) reagent preparation: under a sterile condition, weighing 200mg of cholesterol, and dissolving the cholesterol in 10ml of sterile ultrapure water to prepare a 20mg / ml cholesterol aqueous solution; (2) 1 ml of the mixed solution of the cholesterol aqueous solution and the platelets prepared in the step (1) is injected into the tail vein of the tree shrew, the dosage of the platelets is 3 * 10 < 6 > / ml, the dosage of the cholesterol is 100 micrograms / g, injection is carried out twice, and the time of the second injection is one day away from that of the first injection; a stable tree shrew Alzheimer's disease model can be established after two weeks of first injection. A stable tree shrew AD model can be established two weeks after the first injection, and animals do not die; the tree shrew AD model established by the method is high in modeling rate and small in harm to animals.

Description

Technical Field

[0001] The present invention belongs to the technical field of disease animal model construction, and in particular relates to a method for constructing a tree shrew Alzheimer's disease model. Background Art

[0002] Alzheimer's Disease (AD) is the most common neurodegenerative disease today. Its biological characteristics are the abnormal accumulation of amyloid-β (Aβ) in the brain and the formation of extracellular amyloid plaques and intracellular neurofibrillary tangles formed by hyperphosphorylated microtubule-associated protein (Tau). At present, the annual global expenditure on AD treatment is about 1 trillion US dollars. According to estimates, the cost of AD treatment will double by 2030, which will bring serious economic burden to society and families. However, due to the complex pathological changes of AD and the inconclusive pathogenesis, there is still no good cure in clinical practice, so it is urgent to explore the pathogenesis of AD and develop new drugs to treat AD. Therefore, establishing a good AD disease model is an indispensable part of in-depth research on its etiology, pathological process and prevention and treatment methods.

[0003] AD is a disease caused by multiple causes and with extremely complex pathology. The true cause of AD has not yet been found, so there is no drug that can truly effectively treat it. Scientists have proposed a variety of causes of AD, mainly including the β-amyloid protein cascade hypothesis, the Tau protein hypothesis, neuroinflammation, cholinergic neuron damage, oxidative stress and abnormal glucose metabolism. Based on the above hypotheses, AD animal models are currently divided into three categories: natural aging models, genetic engineering and drug induction. The drug-induced model is usually carried out by injecting β-amyloid protein oligomers, Tau protein fibers, LPS, okadaic acid and other factors that can induce OA symptoms in animals into the brain, but the injection method cannot avoid causing a certain degree of brain damage to the animal, and the fixed injection position leads to a relatively limited range of lesions, which is quite different from the systemic pathological characteristics of AD. Therefore, it is of great significance to establish an animal model with less damage to animals and a larger range of pathogenicity for the study of AD pathology. Summary of the invention

[0004] The purpose of the present invention is to provide a method for constructing a tree shrew Alzheimer's disease model.

[0005] The object of the present invention is achieved by injecting 1 ml of a platelet and cholesterol mixture into the tail vein of a tree shrew, and the platelet dosage is 3×10 6 / ml, cholesterol dose 100μg / g, a total of 2 injections, the second injection time is one day apart from the first injection; a stable tree shrew AD model can be established 2 weeks after the first injection, and no animal deaths; the tree shrew AD model established by this method has a high model success rate and is less harmful to animals.

[0006] The beneficial effects of the present invention are: The modeling cycle is short, and a stable tree shrew AD model can be established 2 weeks after the first injection. If the animals have not died, it means that the animal mortality rate is low. The tree shrew AD model established by this method has a high modeling rate and is less harmful to animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 This is a comparison of the HE staining results in the hippocampus and cortex of tree shrews in the four groups of comparative experiments in this application; Figure 2 This is a comparison chart of immunohistochemical results in the hippocampus and cortex of tree shrews in the four comparative experiments of this application; Figure 3 This is a comparison chart of the Aβ protein expression in the hippocampus and cortex of tree shrews in the four comparative experiments of this application; Figure 4 This is a comparison chart of the expression changes of p-Tau (T231), p-Tau (S396), and Tau proteins in tree shrews in the four comparative experiments of this application; DETAILED DESCRIPTION

[0008] The technical solution of the present invention will be described clearly and completely in combination with the embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, but not all of them. All other embodiments belong to the protection scope of the present invention.

[0009] , Experimental Animals Twenty-four ordinary male Burmese tree shrews weighing 120-150 g were purchased from the Institute of Medical Biology, Chinese Academy of Medical Sciences [SCXK (Yunnan) K2023-0003] and kept in the ordinary animal room of Yunnan Luoyu Biotechnology Co., Ltd. [SYXK (Yunnan) K2021-0003] in stainless steel cages at room temperature (24±2)℃, relative humidity (50±5)%, and light / dark cycle of 12 h / 12 ​​h.

[0010] , main reagents and consumables Reagent name company Cholesterol - Water Soluble Mce Hematoxylin and eosin (HE) staining kit Solebao β-Amyloid Antibodies Abcam Tau Antibody Abcam p-Tau (T231) Antibody Abcam p-Tau (S396) Antibody Abcam GAPDH Antibody Abcam Anti-rabbit IgG CST RIPA Lysis Buffer (Strong) Blue Sky Prestained protein marker Thermo Fisher Bovine serum albumin (BSA) Sigma SurperECLPlus Ultrasensitive Luminescence Prile Sheep serum for blocking Zhongshan Jinqiao Universal two-step test kit Zhongshan Jinqiao DAB colorimetric kit Zhongshan Jinqiao PVDF membrane Millipore 3. Main instruments Instrument Name company Nanophotometer ultra-micro-volume spectrophotometer IMPLEN Protein electrophoresis tank, electrotransfer tank, power supply Bio-Rad ECL Luminescence Imager Tanon Automatic tissue dehydrator Wuhan Junjie Paraffin embedding machine German Leica Rotary microtome German Leica microscope OLYMPUS BX53 4. Preparation of reagents Cholesterol (CHO): Under sterile conditions, weigh 200 mg of cholesterol-water soluble and dissolve it in 10 ml of sterile ultrapure water to prepare a 20 mg / ml cholesterol aqueous solution.

[0011] 5. Experimental methods 5.1 Animal model preparation Tree shrews were randomly divided into the following five groups ①, Normal (NC) group: 1 ml sterile ultrapure water was injected into the tail vein ② Platelet + cholesterol low dose (25 μg / g) group: 1 ml platelet (3×10 6 / ml) and cholesterol (25μg / g) mixture ③, Platelet + cholesterol medium dose (50 μg / g) group: 1 ml platelet (3×10 6 / ml) and cholesterol (50μg / g) mixture ④ Platelet + cholesterol high dose (100 μg / g) group: 1 ml platelet (3×10 6 / ml) and cholesterol (100μg / g) mixture Groups ②-④ were injected with a mixture of platelets and cholesterol of corresponding concentrations through the tail vein according to the experimental groups, once a day. After the first injection, another injection was given every other day, for a total of two times. Platelets and cholesterol needed to be preheated to 37°C before mixing. Group ① was injected with 1 ml of sterile ultrapure water through the tail vein at the same time.

[0012] 5.2 Tree shrew blood collection and platelet preparation methods The tree shrew was anesthetized by intraperitoneal injection of 3% sodium pentobarbital at 2μl / g body weight. After complete anesthesia, the tree shrew was held in the hand, and the blood collector held the back of the tree shrew's neck tightly from the back with the thumb and index finger of the left hand and laid it flat on the operating table. When collecting blood, the thumb and index finger of the left hand gently pressed the skin on both sides of the tree shrew's ears, so that the eyeballs protruded slightly and the retroorbital venous plexus was congested. A 3-4 cm long hard glass siphon (capillary inner diameter 0.5-1.0 mm) was held in the right hand, and the blood collector was made at a 45° angle to the tree shrew's face. The blood was inserted from the inner corner of the eye, with the oblique surface facing the eyeball and the insertion surface facing the posterior border of the orbit. The insertion depth was about 4-5 mm. When resistance was felt during rotation, the advancement was stopped, and a little blood was gently pushed outward to allow it to flow out of the siphon naturally, and then the head was slightly tilted to allow the blood to flow into the centrifuge tube. After the blood collection is completed, release the tree shrew's head with your left hand, pull out the blood collector, and press the puncture hole at the corner of the eye with a dry cotton ball to prevent bleeding. The left and right eyes can be rotated for blood collection, and the blood collection dose is 1 ml each time.

[0013] Place the centrifuge tube containing blood in a centrifuge and centrifuge at 22℃ 200r / min for 10 minutes. Take the upper platelet-rich plasma in a clean bench and put it into another centrifuge tube; centrifuge at 22℃ 1500r / min for 15 minutes and discard the supernatant. The sediment at the bottom of the tube is platelets. Wash it three times with platelet washing solution to obtain purified platelets.

[0014] Tail vein injection method for tree shrews The tree shrew is fixed in a restraining bag, the tail is exposed, and the net mouth is tied tightly; the ventral side faces up, and the fur on the ventral median of the tree shrew's tail is cut off with clippers to fully expose the skin and blood vessels of the tree shrew's tail. The area is wiped with an alcohol cotton ball, and then wiped dry with a dry cotton ball to expand the tail vein for easy injection. The tip of the tree shrew's tail is controlled by the right hand, and the index finger and middle finger of the left hand clamp the base of the tree shrew's tail on the dorsal and ventral sides and push toward the tip of the tail. Stop and clamp the tree shrew's tail when the blood vessels at the injection site are clearly visible. The thumb, ring finger and little finger of the left hand control the tip of the tree shrew's tail, and the right hand holds a 1ml disposable syringe (No. 4 needle) with the needle tip beveled upward, so that the needle is parallel to the vein (less than 15°) and slowly inserts the needle from the end of the tail to the proximal end. The depth of the needle should be more than 1 / 2. Slightly withdraw the syringe with the right hand. When blood returns, it proves that the tail vein is punctured successfully. Push gently without resistance to start the injection.

[0015] Behavioral observation of tree shrews After the injection, the diet, exercise, coat color and mental state of each group of tree shrews were observed daily.

[0016] 5.5 HE staining After the administration of the drug, the tree shrews were kept normally for 2 weeks and then the tree shrews were collected. After the tree shrews were anesthetized, physiological saline and 4% paraformaldehyde solution were perfused through the heart, and the brain was taken and placed in 4% paraformaldehyde to prepare paraffin sections for subsequent HE and immunohistochemical staining.

[0017] HE staining: paraffin sections were dewaxed to water and stained with hematoxylin at room temperature for 3-5 minutes; washed with tap water for 10 minutes to remove floating color, differentiated with 1% hydrochloric acid alcohol for 2 seconds; rinsed with tap water for 10 minutes, observed the cell nucleus to be blue-purple under a microscope, and then stained with eosin at room temperature for 5 minutes; dehydrated in gradient alcohol, transparent in xylene, sealed with neutral gum, observed the color development results under an ordinary optical microscope and photographed.

[0018] Immunohistochemistry to detect Aβ expression Paraffin sections were dewaxed to water, and sections were immersed in an antigen repair box with pH=6.0 citrate buffer in a microwave oven for antigen repair. 3% hydrogen peroxide was used to block the sections in the dark for 30 minutes (to eliminate the activity of endogenous peroxidase); 5% goat serum was used to block the sections for 1 hour, and then the primary antibody (Aβ1:400) was added and incubated overnight at 4°C. After rinsing with PBST, the universal two-step detection reagent was added and incubated at room temperature for 30 minutes; after rinsing with PBST, the freshly prepared DAB color development solution was added and incubated at room temperature in the dark for 5-15 minutes. The color development was terminated with distilled water; hematoxylin was used for counterstaining, 1% hydrochloric acid ethanol was used for differentiation, and the sections were routinely dehydrated, transparent, and sealed with neutral gum. The color development results were observed under an ordinary optical microscope and photographed.

[0019] Detection After the administration of the tree shrews, they were kept normally for 2 weeks and then collected. After the tree shrews were anesthetized, the chest was opened and the heart was perfused with normal saline. After the liver turned white, the tree shrew brain tissue was removed, the brain was separated on ice, and gently rinsed with 0.1mol / L PBS. 500μL of RIPA lysis buffer (containing 50μL protease inhibitor) was added for every 100mg of fresh tissue. After the tissue was fully minced with sterile ophthalmic scissors, it was homogenized with an ultrasonic cell disruptor for 20s in an ice bath environment, and then left to lyse for 20min on an ice bath. Centrifuge at 4℃, 12000rpm for 10min, collect the supernatant, aspirate about 360μL of supernatant in each tube, add 90μL 5× loading buffer (1 / 4 of the supernatant volume), mix thoroughly, centrifuge for 10s, and boil in a 100℃ metal bath for 10min to fully denature the protein. After the sample cooled, measure the protein concentration. Then, SDS-PAGE protein electrophoresis was performed, using 80V constant voltage electrophoresis until the lower edge of the sample touched the separation gel (about 30 minutes), and then changing to 130V constant voltage to continue electrophoresis for nearly 2 hours, until the bromophenol blue just overflowed the lower edge of the separation gel and stopped electrophoresis; wet transfer was performed at 280mA / 1h, and 5% skim milk was added for blocking at room temperature for 2h, and primary antibodies (rabbit anti-Tau (phospho T231) antibody 1:1000, rabbit anti-Tau (phospho S396) antibody 1:1000, rabbit anti-Tau antibody 1:1000, GAPDH antibody 1:2000) were added and incubated overnight in a silent mixer in a 4℃ refrigerator, and the membrane was washed 3 times with TBST buffer, incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:1000) at room temperature for 2h, and washed 3 times with TBST buffer. ECL reagent was added using the chemiluminescence method and developed on a gel imaging system.

[0020] , Experimental results 6.1 Behavioral observations of tree shrews The activities of tree shrews in the NC group were normal and there were no abnormalities. Compared with the NC group, the tree shrews in the 100μg / g group were listless and their activities decreased 2 days after the first injection. As time went on, the activities of tree shrews further decreased and the glossiness of their hair became worse. The activities of tree shrews in the 50μg / g group decreased 5 days after the first injection compared with the previous few days. The activities of tree shrews in the 25μg / g group were normal and there were no abnormalities.

[0021] Staining results Under the optical microscope, the morphological structure of the neurons in the hippocampus of the tree shrews in the NC group was complete, neatly arranged, with abundant cytoplasm and clear nuclei; a small number of neurons in the hippocampus of the tree shrews in the 25μg / g group showed nuclear pyknosis; most neurons in the hippocampus of the tree shrews in the 50μg / g group showed nuclear pyknosis; most neurons in the hippocampus of the 100μg / g group showed obvious changes in morphological structure, with increased vacuoles, irregular arrangement of cells, visible nuclear pyknosis, unclear boundaries between cytoplasm and nucleus, and the disappearance of the nucleus. Figure 1 shown.

[0022] Immunohistochemistry to detect Aβ expression In order to explore the damage caused by platelets combined with cholesterol to the brain of tree shrews, we used immunohistochemistry to detect the expression of Aβ protein in the brain of tree shrews. The results showed that compared with the NC group, the injection of platelet and cholesterol mixture increased the expression of Aβ in the hippocampus and cortex of the tree shrew brain, and with the increase of cholesterol dose, the expression of Aβ also increased. The 50μg / g group and the 100μg / g group were statistically different from the NC group (p < 0.05), and the 100μg / g group was statistically different from the 50μg / g group (p < 0.05).

[0023] Detection Western bolt was used to measure the protein levels of Tau, p-Tau (T231) and p-Tau (S396) in the brain tissue of tree shrews. The results showed that compared with the NC group, the p-Tau (T231) level in the brain of tree shrews after injection of platelet and cholesterol mixture was significantly increased, and the difference between the 50μg / g group and the 100μg / g group and the NC group was statistically significant (p < 0.05); compared with the NC group, the p-Tau (S396) level in the brain of tree shrews after injection of platelet and cholesterol mixture was significantly increased, and the difference between the 50μg / g group and the 100μg / g group and the NC group was statistically significant (p < 0.05), and the difference between the 100μg / g group and the 50μg / g group was statistically significant (p < 0.05); while there was no significant difference in Tau protein level (p > 0.05). Table 1 Changes in the expression of p-Tau (T231), p-Tau (S396) and Tau proteins in the four groups of tree shrews during the experiment Experimental Grouping p-Tau (T231) / GAPDH p-Tau (S396) / GAPDH Tau / GAPDH NC 0.528±0.168 0.35±0.113 0.558±0.054 25μg / g 0.663±0.226 0.676±0.215 0.536±0.025 50μg / g <![CDATA[0.866±0.173 a ]]> <![CDATA[0.895±0.186 b ]]> 0.541±0.043 100μg / g <![CDATA[1.025±0.107 b ]]> <![CDATA[1.252±0.167 b,c ]]> 0.57±0.112 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 50μg / g group.

Claims

1. A method for constructing a tree shrew Alzheimer's disease model, characterized in that: The steps include: (1) Reagent preparation: Under sterile conditions, weigh 200 mg of cholesterol and dissolve it in 10 ml of sterile ultrapure water to prepare a 20 mg / ml cholesterol aqueous solution; (2) 1 ml of the mixed solution of cholesterol aqueous solution prepared in step (1) and platelets was injected into the tail vein of the tree shrew, with a platelet dose of 3×10 6 / ml, cholesterol dose 100μg / g, 2 injections in total, the second injection time is one day apart from the first injection; a stable tree shrew Alzheimer's disease model can be established 2 weeks after the first injection.

2. The method for constructing a tree shrew Alzheimer's disease model according to claim 1, characterized in that: The method for preparing platelets in step (2) is as follows: The tree shrews were anesthetized by intraperitoneal injection of 3% sodium pentobarbital at 2 μl / g body weight. After complete anesthesia, the tree shrews were held in the hand. The blood collector used the thumb and index fingers of his left hand to pinch the back of the tree shrew's neck from the back and laid it flat on the operating table. When drawing blood, the thumb and index finger of his left hand gently pressed the skin on both sides of the tree shrew's ears to make the eyeballs protrude slightly and the retroorbital venous plexus congested. A 3-4 cm long hard glass siphon was held in the right hand, and the blood collector was placed at a 45° angle to the tree shrew's face. Insert the needle into the tree shrew corner with the bevel facing the eyeball and the insertion surface facing the posterior border of the orbit. The insertion depth is 4 to 5 mm. When you feel resistance when rotating, stop pushing. Gently push a little blood outwards and the blood will naturally flow out of the siphon tube. Then tilt the head slightly to allow the blood to flow into the centrifuge tube. After the blood collection is completed, release the tree shrew's head with your left hand and pull out the blood collector at the same time. Press the puncture hole in the corner of the eye with a dry cotton ball to prevent bleeding. The left and right eyes can be rotated for blood collection. The blood collection dose is 1 ml each time. Place the centrifuge tube containing blood in a centrifuge and centrifuge at 22℃ 200r / min for 10 minutes. In the clean bench, draw the upper platelet-rich plasma into another centrifuge tube; centrifuge at 22℃ 1500r / min for 15 minutes and discard the supernatant. The sediment at the bottom of the tube is platelets, which should be washed three times with platelet washing solution to obtain purified platelets.

3. The method for constructing a tree shrew Alzheimer's disease model according to claim 1, characterized in that: The tail vein injection method is specifically: The tree shrew was fixed in a restraining bag, the tail was exposed, and the net was tied tightly; with the ventral side facing up, the fur on the ventral median of the tree shrew's tail was cut off with clippers to fully expose the skin and blood vessels of the tree shrew's tail, and this part was wiped with an alcohol cotton ball, and then dried with a dry cotton ball to expand the tail vein for easy injection. The tip of the tree shrew's tail was controlled with the right hand, and the base of the tree shrew's tail on the dorsal and ventral sides was clamped with the index and middle fingers of the left hand and pushed toward the tip of the tail. When the blood vessels at the injection site were clearly visible, the tail was stopped and clamped. The tip of the tree shrew's tail was controlled with the thumb, ring finger and little finger of the left hand. A 1 ml disposable syringe was held in the right hand with the needle tip beveled upward, so that the needle was parallel to the vein and slowly inserted from the tail end to the proximal end. The needle insertion depth was more than 1 / 2 of the needle. The syringe was slightly withdrawn with the right hand. When blood returned, it proved that the tail vein was punctured successfully. The injection could be started by gently pushing without resistance.

4. The method for constructing a tree shrew Alzheimer's disease model according to claim 1, characterized in that: The tree shrews are kept at room temperature of 24±2°C, relative humidity of 50±5%, and a light / dark cycle of 12h / 12h.

Images