Construction method of Alzheimer's disease mouse model

Abstract

The invention discloses a construction method of an Alzheimer's disease mouse model, which comprises the following steps: hybridizing an ApoE4 (+ / +) transgenic mouse with an APP / PSEN1 double transgenic mouse, and identifying the obtained filial generation; and hybridizing the first filial generation with the genotype of ApoE4 (+ / -) / APP / PSEN, and identifying the obtained second filial generation to obtain the homozygous positive ApoE4 (+ / +) / APP / PSEN1 Alzheimer's disease mouse model. Through behavioral, morphological and molecular biological detection, the ApoE4 (+ / +) / APP / PSEN1 mouse conforms to the characteristic phenotype of the Alzheimer's disease in the aspects of cognitive function, brain tissue pathology and molecular markers, and can be used as an animal model of the Alzheimer's disease.

Description

Technical Field

[0001] The present invention belongs to the technical field of model construction, and specifically discloses a method for constructing a mouse model of Alzheimer's disease. Background Art

[0002] Alzheimer's Disease (AD) is a progressive neurodegenerative disease, which is mainly manifested as memory loss, cognitive dysfunction and dementia. Animal models play an important role in studying the pathological mechanism of Alzheimer's disease and evaluating potential treatment methods.

[0003] The APP / PSEN / TAU triple transgenic mouse model can reproduce two major pathological features of Alzheimer's disease - amyloid plaques and neurofibrillary tangles. This model obtains pathological changes of neurofibrillary tangles by introducing a mutant tau gene. However, in fact, the neurofibrillary tangles in Alzheimer's disease are not caused by tau gene mutations, but by other inducing factors. In addition, research shows (Aida Attar, PLOS ONE) that when evaluating the cognitive defects of APP / PSEN / TAU triple transgenic mice, no significant cognitive defects were found in mice using the standard Barnes maze training protocol (15 training trials).

[0004] In summary, it is of urgent practical significance to newly develop an animal model that can cover the pathological changes of Alzheimer's disease, meet the risk factors for onset, and at the same time have cognitive functions consistent with the characteristic phenotypes of Alzheimer's disease. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for constructing a mouse model of Alzheimer's disease, including: hybridizing ApoE4(+ / +) transgenic mice with APP / PSEN1 double transgenic mice, and identifying the obtained offspring; using the first-generation offspring with the genotype ApoE4(+ / -) / APP / PSEN for hybridization, and identifying the obtained second-generation offspring to obtain a homozygous positive ApoE4(+ / +) / APP / PSEN1 Alzheimer's disease mouse model.

[0006] Preferably, the identification of the obtained second-generation mice includes identifying the ApoE4(+ / +) / APP / PSEN1 mice according to the molecular, pathological and behavioral characteristics of Alzheimer's disease.

[0007] Preferably, the method for preparing the ApoE4(+ / +) transgenic mice comprises: injecting a human ApoE4 transgenic fragment into a mouse fertilized egg by microinjection technology; transplanting the surviving fertilized eggs into pseudopregnant female mice to obtain F0 generation mice; mating the F0 generation mice with wild-type mice to obtain F1 generation heterozygous mice; mating the F1 generation heterozygous mice with each other to obtain F2 generation mice, and screening to obtain the ApoE4(+ / +) transgenic mice.

[0008] The mouse model of Alzheimer's Disease (AD) is an essential tool for studying the pathogenesis and treatment strategies of AD. Currently, the best method for establishing an AD model is transgenic technology. The existing transgenic models of Alzheimer's disease mainly transfer the mutant genes in familial AD (FAD), and the most commonly used ones are the mutant genes of β-amyloid precursor protein (APP) and presenilin (PSEN).

[0009] ApoE4 was the first identified Alzheimer's disease risk gene reported in 1993 (Serrano-Pozo A, Das S, Hyman B T. APOE and Alzheimer's disease: advances in genetics, pathophysiology, and therapeutic approaches. Lancet Neurol, 2021, 20(1): 68-80.). The allele frequency of ApoEε4 is 15% in the total population, but up to 40% in AD patients. The AD onset risk of ApoEε4 homozygous individuals is 12 times that of non-carriers, and the onset age also decreases significantly (Lane-Donovan C, Herz J. ApoE, ApoEReceptors, and the Synapse in Alzheimer's Disease. Trends Endocrinol Metab, 2017, 28(4): 273-284).

[0010] There are many types of AD model mice, but AD model mice carrying ApoE4 at the same time are not common, which limits the research exploration on the relationship between genetic background and disease occurrence, and brings difficulties to the further development of directions such as the exploration of the pathogenesis of Alzheimer's disease, the discovery of new drug targets, and preclinical efficacy evaluation. In addition, it is difficult to directly carry out population intervention studies on individuals with different ApoE4 genotypes, and it is not easy to obtain brain tissue samples. It is impossible to directly observe the intervention effect and pathological changes, and only by collecting body fluids for index detection can indirectly reflect individual changes. To solve this technical problem, constructing ApoE4 genotype Alzheimer's disease model mice can carry out etiological and intervention studies after obtaining brain tissue samples, explore the mechanisms related to the occurrence and development of Alzheimer's disease, and lay a foundation for the discovery of new treatment targets for Alzheimer's disease, drug research and development, and preclinical efficacy evaluation.

[0011] The present invention uses transgenic technology to replace the tauP301L mutant gene in the APP / PSEN / TAU triple transgenic model with the ApoE4 gene, and establishes an ApoE4 genotype Alzheimer's disease mouse model, and this transgenic mouse conforms to the pathological state of Alzheimer's disease. Through behavioral, morphological, and molecular biological detections, ApoE4(+ / +) / APP / PSEN1 mice are in line with the characteristic phenotypes of Alzheimer's disease in terms of cognitive function, brain tissue pathology, and molecular markers, and can be used as an animal model of Alzheimer's disease. And the ApoE4 genotype Alzheimer's disease mouse is an animal model that can not only cover the pathological changes of Alzheimer's disease but also conform to the risk factors of the disease. This model can be used for in vivo gene function analysis, disease pathogenesis exploration, new drug target discovery, and preclinical efficacy evaluation and other studies. Brief Description of the Drawings

[0012] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0013] Figure 1 Shows the construction strategy of ApoE4(+ / +) humanized mice provided according to an embodiment of the present invention.

[0014] Figure 2 Shows the expression level of exogenous ApoE4 alleles provided according to an embodiment of the present invention; where M: DNA marker; WT: wild type; HE: heterozygote; HO: homozygote. Figure 2 By detecting the expression level of the exogenous ApoE4 transgenic fragment, the total ApoE gene expression level of ApoE4(+ / +) transgenic mice is detected by RT-PCR method to identify ApoE4(+ / +) transgenic mice.

[0015] Figure 3 It shows a schematic diagram of the method for obtaining ApoE4(+ / +) / APP / PS1 transgenic mice by hybridization according to an embodiment of the present invention; the ApoE4(+ / +) transgenic mice are hybridized with APP / PSEN1 mice to obtain the first-generation mice with the genotype of ApoE4(+ / -) / APP / PSEN, and then they are hybridized again. After screening, the triple-positive second-generation mice, that is, ApoE4(+ / +) / APP / PSEN1 transgenic mice, are obtained.

[0016] Figure 4 It shows the identification results of the genotypes of the hybrid offspring according to an embodiment of the present invention.

[0017] Figure 5 It shows the detection results of the escape latency in the first 4 days before the water maze experiment training according to an embodiment of the present invention.

[0018] Figure 6 It shows the latency of each group of mice to reach the platform on the 5th day of the water maze experiment according to an embodiment of the present invention.

[0019] Figure 7 It shows the time of each group of mice in the target quadrant on the 5th day of the water maze experiment according to an embodiment of the present invention.

[0020] Figure 8 It shows the number of times each group of mice passed through the platform on the 5th day of the water maze experiment according to an embodiment of the present invention.

[0021] Figure 9 It shows the detection of senile plaques in the mouse brain tissue by Aβ42 immunohistochemistry. The pathological manifestations of senile plaques in the brain tissue of ApoE4(+ / +) / APP / PS1 transgenic mice are detected by immunohistochemistry. The results show that a large number of senile plaques appear in the cerebral cortex of ApoE4(+ / +) / APP / PS1 transgenic mice.

[0022] Figure 10 It shows the detection of amyloid deposition in the mouse brain tissue by Congo red staining. The results show that the amyloid deposition in the cerebral cortex, hippocampal CA1 and CA3 regions of ApoE4(+ / +) / APP / PS1 transgenic mice is more in quantity and larger in area.

[0023] Figure 11 It shows the detection of the cell morphology of the brain tissue by HE staining. The results show that the number of neurons decreases in the cortex and hippocampal regions of ApoE4(+ / +) / APP / PS1 transgenic mice, and pathological phenomena such as nuclear pyknosis and cell degeneration occur.

[0024] Figure 12It shows the detection of the total tau content in the mouse cerebral cortex tissue by the Western blot method according to an embodiment of the present invention. *: P < 0.05 compared with C57 mice; #: P < 0.05 compared with APP / PS1 mice.

[0025] Figure 13 It shows the detection of the p-tau content in the mouse cerebral cortex tissue by the Western blot method according to an embodiment of the present invention. The expression levels of different forms of tau protein, namely total Tau protein and p-Tau, were detected by the Western blot method in the brain tissues of ApoE4(+ / +) / APP / PS1 transgenic mice. The results showed that the expression of total Tau protein in the brain tissues of ApoE4(+ / +) / APP / PS1 transgenic mice was significantly higher than that in the other three genotypes of mice. However, the expression level of the pathological form p-Tau of Tau protein did not increase among the four types of mice. This indicates that ApoE4(+ / +) / APP / PS1 transgenic mice possess the tau pathological characteristics of Alzheimer's disease and are superior to APP / PS1 mice and ApoE4(+ / +) mice in terms of tau pathology manifestation.

[0026] Figure 14 It shows the detection of the APP content in the mouse cerebral cortex tissue by the Western blot method according to an embodiment of the present invention. The results showed that the APP content in the brain tissues of ApoE4(+ / +) / APP / PS1 transgenic mice was significantly higher than that in wild-type mice and ApoE4(+ / +) mice, indicating that ApoE4(+ / +) / APP / PS1 transgenic mice possess the Aβ pathological characteristics of Alzheimer's disease. Detailed implementation manners

[0027] To make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the following further describes the present invention in detail with reference to specific embodiments and the accompanying drawings. It should be noted that the following embodiments do not limit the scope of protection required by the present invention and are only illustrative embodiments. The experimental methods used in the following embodiments are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0028] Example 1: Construction of ApoE4(+ / +) transgenic mouse model

[0029] According to the cDNA sequence of the human ApoE4 gene (Gene ID: 348), specific primers were designed, and restriction enzyme sites (such as EcoRI and BamHI) were introduced at the 5' and 3' ends of the primers for subsequent cloning. Using the genome of ApoE knockout mice as a template, PCR amplification was performed with the primers to obtain the cDNA fragment of the hApoE4 gene.

[0030] PCR conditions: Pre-denaturation: 94°C, 3 minutes; Cycling: 94°C for 30 seconds, 62°C for 35 seconds, 72°C for 35 seconds, for a total of 35 cycles; Final extension: 72°C, 5 minutes.

[0031] The PCR system is shown in Table 1 below:

[0032]

[0033]

[0034] The PCR product and the vector plasmid (such as pBluescript) were double digested with restriction enzymes (such as EcoRI and BamHI). The digested hApoE4 cDNA fragment was inserted into the vector plasmid to construct a recombinant vector. The fragment was ligated to the vector through a ligase reaction and transformed into Escherichia coli (such as DH5α), and positive clones were screened and verified by sequencing.

[0035] The recombinant vector was linearized with restriction enzymes to obtain a transgenic fragment containing hApoE4 cDNA. The linearized fragment was recovered by gel electrophoresis and a purification kit (such as Qiagen Gel Extraction Kit) for microinjection.

[0036] The purified linearized transgenic fragment was injected into the pronucleus of C57BL / 6 mouse fertilized eggs by microinjection technology. The injected fertilized eggs were cultured in M16 medium until the 2-cell stage. The surviving fertilized eggs were transplanted into the ampulla of the oviduct of pseudopregnant female mice. Pseudopregnant female mice were induced to be in a pseudopregnant state by mating with vasectomized male mice. Four weeks after the pups were born, 1-2 mm of tail tip tissue was cut and genomic DNA was extracted. The integration of the exogenous ApoE4 gene was detected by RT-PCR method.

[0037] The appearance of an amplification product at the 576 bp position indicated that the ApoE4 gene was successfully integrated and was homozygous (+ / +). Figure 1 Schematic diagram of the E4(+ / +) transgenic fragment structure, showing the connection mode of hApoE4 cDNA and the vector plasmid. Figure 2 Electrophoresis diagram of the RT-PCR result, showing an obvious amplification band at 576 bp, proving the successful construction of the ApoE4(+ / +) mouse model.

[0038] Example 2: Generation of ApoE4(+ / +) / APP / PS1 triple transgenic mice

[0039] The ApoE4(+ / +) transgenic mice constructed in Example 1 were crossed with APP / PSEN1 double transgenic Alzheimer's disease model mice. The APP / PSEN1 double transgenic mice can be purchased from Jackson Laboratory in the United States. First-generation cross: ApoE4(+ / +) mice were mated with APP / PSEN1 mice to obtain first-generation (F1) heterozygous ApoE4(+ / -) / APP / PS1 mice. Second-generation cross: The F1 heterozygous mice were mated with each other to obtain second-generation (F2) homozygous ApoE4(+ / +) / APP / PS1 mice.

[0040] Mouse tail tissues of 2 - 3 mm were cut, and DNA was extracted using a genomic DNA extraction kit (such as Qiagen DNeasy Kit). Specific primer pairs were used to perform PCR amplification on the APP, PSEN1, and ApoE4 genes. The PCR products were subjected to agarose gel electrophoresis to detect the target bands. When analyzing the results, the ApoE4(+ / +) / APP / PS1 triple transgenic mice should simultaneously show the specific bands of ApoE4, APP, and PSEN1. Figure 3 It is a schematic diagram of the hybridization strategy, showing the hybridization process of ApoE4(+ / +) mice and APP / PSEN1 mice and the genotype distribution of the offspring. Table 2 shows the primer sequences of the APP and PSEN1 genes.

[0041] All transgenic mice were raised in a SPF-class animal house, maintaining a 12-hour light / dark cycle and free access to food and water. Behavioral tests (such as Morris water maze) and pathological analyses (such as detection of β-amyloid deposition) were performed on ApoE4(+ / +) / APP / PS1 mice to verify their Alzheimer's disease phenotype. This model can be used to study the mechanism of action of Alzheimer's disease, as well as to screen and evaluate potential therapeutic drugs.

[0042] The ApoE4 primers are shown in Table 2 below:

[0043]

[0044] The APP / PSEN1 primers (provided by the Jackson National Animal Center in the United States) are shown in Table 3 below:

[0045]

[0046] Example 3: Cognitive function characteristics of ApoE4 genotype Alzheimer's disease mice

[0047] The Morris water maze is a classic experiment for detecting the hippocampal cognitive function of Alzheimer's disease. The learning, memory, and spatial cognitive abilities of ApoE4(+ / +) / APP / PS1 transgenic mice are evaluated through behavioral experiments in the water maze.

[0048] The water maze used for mouse detection has a diameter of 1.5 m. Before detecting AD model mice, an appropriate amount of edible titanium dioxide needs to be added to the pool to increase the contrast between the animals and the background; the platform has a diameter of 10 cm and the platform plane is 2 cm underwater; markers of different colors and shapes are pasted on the side walls of the water maze to help the mice remember the relative position of the platform; the indoor lights are avoided from directly shining on the water surface; the camera is above the center of the pool, and the camera focus and clarity are adjusted to identify the movement state of the mice. The camera is connected to a computer, and the tracking system of the camera is used to record and analyze the movement trajectory of the mice.

[0049] The mouse water maze experiment is divided into two stages: the training stage and the probe test. In the training stage, the mice need to be trained continuously for 4 days so that the mice can remember the position of the platform through the prompt of the surrounding markers. During these 4 days, the mice need to be put into the water from the quadrant opposite the platform every day for testing. When the mice are put into the water against the side wall of the pool, keep the back of the mice facing the side wall of the pool, and at the same time start the software and record for 90 s. If the mice climb onto the platform and stay for 5 s, it is considered that the mice can successfully find the platform, and the software timing is stopped. If the mice do not successfully find the platform after 90 s, an experimenter guides the mice to find the platform in the water and stays for 1 min. The probe test is carried out on the 5th day, and the start time of the probe test should be more than 24 h after the last mouse training time. Remove the platform in the water, put the mice into the water from the quadrant opposite the platform, time for 90 s, then pick up the mice, dry their hair and put them back into the cage.

[0050] The environment needs to be kept quiet during the experiment. During the training experiment of the mice, the time from entering the water to climbing onto the platform is the latency. During the probe test, the number of times the mice shuttle through the original platform position is recorded as the "number of times through the platform".

[0051] It has been verified that the learning and spatial memory abilities of ApoE4(+ / +) / APP / PS1 transgenic mice are significantly lower than those of wild-type mice and APP / PS1 mice, and there is also a downward trend compared with E4(+ / +) mice.

[0052] Results: The escape latency of each group of mice before the 4-day training in the water maze experiment is as Figure 5 shown, the latency of each group of mice to reach the platform on the 5th day of the water maze experiment is as Figure 6 shown, the activity time of each group of mice in the target area on the 5th day of the water maze experiment is as Figure 7 shown, and the number of times each group of mice passes through the platform on the 5th day of the water maze experiment is as Figure 8As shown in the figure. It can be seen from the above results that the learning and spatial memory abilities of ApoE4(+ / +) / APP / PS1 transgenic mice are significantly reduced compared with those of wild-type and APP / PS1 mice, and there is also a downward trend compared with E4(+ / +) mice.

[0053] The Barnes maze was originally designed by Carol Barnes for rat experiments to avoid the stress response caused by swimming in the Morris water maze (MWM), and then it was improved to be applicable to mice. The basic principle of the Barnes maze test is to let mice find a hidden dark escape box under the drive of negative reinforcement factors such as bright light, open environment, and noise. Compared with the Morris maze, the Barnes maze can be used to evaluate spatial reference memory and learning, and mice have more exploratory behaviors. The Barnes maze is widely used for the assessment of spatial memory in Alzheimer's disease mouse models.

[0054] In the training stage, the learning ability is evaluated by the time (latency) for mice to find the target hole and the number of holes searched incorrectly (holes searched, HS). In the probe test stage, long-term memory is usually evaluated by the time mice stay in the target quadrant or the number of holes searched.

[0055] The maze used in the experiment is made of 13-mm thick white PVC board, with a diameter of 48 inches. Twenty holes with a diameter of 1.75 inches are evenly distributed on the periphery, and the hole is 1 inch away from the edge. The maze is installed on a 35-inch high rotating stool to keep balance. The escape box is a modified mouse cage, placed 1.25 inches below the target hole. The escape platform is made by transforming a square petri dish, and the ramp is made of laminated cardboard and cleaned with 70% ethanol. Black paper is pasted on the outside of the cage to keep the inside dark, so as to attract mice to enter. A separate test room is specially set up in the laboratory, and eight brightly colored graphics (squares, triangles, circles) are installed around the room as visual cues. In addition, the asymmetrical layout of the room also provides additional environmental cues for mice. During the experiment, after each test, the area around the target hole or the whole maze is cleaned with 70% ethanol to prevent odor interference. At the same time, after testing every 3 mice, the maze is rotated clockwise to prevent mice from using the visual or odor cues of the maze itself for positioning. All experiments are recorded using a COP Security Monochrome CCD camera and MyTV / x software.

[0056] The test of mice in the Barnes maze is divided into three stages: adaptation period (1 day), training period (4 days), and probe test (1 day).

[0057] Adaptation period: Before the experiment began, each mouse was required to acclimatize to the laboratory for 1 hour. Subsequently, 2 - 4 mice from the same cage were placed separately in individual waiting cages until they completed all the tests. This treatment prevented individual mice from experiencing stress due to being left alone in their home cages and avoided the behavior of tested mice affecting untested mice. During the adaptation period, the mouse was placed in the center of the maze and covered with a transparent 3,500 ml glass beaker for 30 seconds while white noise was played. Subsequently, the researcher slowly guided the beaker (10 - 15 seconds) to the target hole, allowing the mouse to enter the escape box. The mouse had 3 minutes to enter the escape box on its own. If it failed to enter within the specified time, the researcher would gently nudge it into the box with the beaker. Ensuring the mouse successfully entered the escape box was a crucial step, which made the mouse aware of the existence of the escape box and familiar with the way to enter. After entering the escape box, the mouse stayed there for 1 minute and was then sent back to the waiting cage. After all the mice completed the adaptation period, they were sent back to their home cages.

[0058] Training period: During the training period, each mouse was first placed in an opaque cylindrical cardboard cover that was 10 inches high and 7 inches in diameter for 15 seconds. Subsequently, the cover was removed and the buzzer was turned on while the mouse explored the maze for 2 minutes. If the mouse found the target hole and entered the escape box within the specified time, it could stay for 1 minute and then return to the waiting cage. If it failed to enter the target hole, the researcher would guide it to the target hole with the beaker and let it enter on its own, allowing up to 3 minutes. If it still did not enter, the researcher would manually place it in the escape box and send it back to the waiting cage after 1 minute. During the test, the buzzer was turned off immediately after the mouse entered the escape box. The training protocol included 15 training trials (3 trials on the first day and 4 trials each on the second - fourth days). The training of each mouse usually took 5 - 7 minutes, and 4 mice were trained simultaneously with an interval of 20 - 30 minutes between each group.

[0059] During the training phase, the experimenter recorded:

[0060] Primary latency: The time required for the mouse to first identify the target hole (Note: The mouse does not necessarily enter immediately after identifying the target hole).

[0061] Holes Searched, HS: The number of times the mouse sniffed or poked its head at all the holes.

[0062] Primary HS: The number of times the mouse explored other holes before first identifying the target hole.

[0063] All data were recorded by blinded experimenters to avoid subjective influence. In addition, approximately 70% of the data were randomly rechecked by a second blinded experimenter to ensure data accuracy. After comparing the data of the two experimenters, no significant differences were found, and all data were analyzed as the mean value.

[0064] Probe test: In the probe test on the last day, the escape box was removed. After the mouse was placed in the cardboard cover in the center of the maze for 15 seconds, the cover was removed, the buzzer sounded, and the mouse had 2 minutes to freely explore the maze. During this process, the residence time of the mouse in the target quadrant and the number of holes searched (HS) were recorded.

[0065] With a 15 - trial training protocol, no obvious cognitive differences were found between APP / PSEN / TAU triple - transgenic mice and wild - type mice. In the first 4 trials, there were significant differences in the latency of the two groups of mice, but after the 5th trial, the differences disappeared. There were obvious cognitive differences between ApoE4(+ / +) / APP / PS1 transgenic mice and wild - type mice, and these differences did not disappear after the 5th trial.

[0066] Example 4: Morphological characteristics of the brain tissue of ApoE4 - genotype Alzheimer's disease mice

[0067] Plaque pathology (Aβ accumulation, senile plaques) and tangle pathology (tau protein accumulation, neurofibrillary tangles) are classical pathological evaluation indicators for Alzheimer's disease. The pathological manifestations of the above indicators in the brain tissues of ApoE4(+ / +) / APP / PS1 transgenic mice were detected by immunohistochemistry and pathological staining methods. After the behavioral detection experiment, the experimental animals were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution (10 mg / ml). After the operating table was set up, the abdominal skin and muscle were incised under the xiphoid process of the sternum, the diaphragm was cut open, and the ribs cut and turned up from both sides of the chest were fixed with hemostatic forceps to fully expose the heart. A puncture needle was inserted into the ascending aorta, the needle was fixed, and the right auricle was cut open at the same time. Ice-cold normal saline was rapidly injected with a 50-ml syringe until no bloody fluid flowed out of the right auricle and the liver turned yellowish-brown. Immediately afterwards, to complete the fixation process, the mice were perfused with 200 ml of 4% paraformaldehyde in PBS (pH 7.2 - 7.4, 4°C) until the liver became hard and the limbs became stiff. After quickly decapitating the mice and removing the brain, the hippocampal region was taken and 3-mm coronal sections were cut front and back, marked, and then placed in a 4°C solution of 20% sucrose in paraformaldehyde for more than 24 h for fixation. Gradient alcohol dehydration was carried out after the front and back fixation. At 30-min intervals, starting from the low concentration of 50%, gradient alcohol dehydration was carried out in the following order (70% → 80% → 95% I → 95% II → 100% ethanol I → 100% ethanol II). After the dehydration process was completed, the samples were cleared twice with xylene at 20-min intervals, and then infiltrated with wax and embedded. The glass slides used for section preparation were soaked in warm soapy water for 2 - 4 hours before sectioning, then rinsed thoroughly with tap water, dried in an oven at 70°C before acid treatment, rinsed again with tap water, then rinsed three times with distilled water and dried again in a 70°C oven. After the dried sections were cooled, they were soaked in 95% ethanol, and the glass slides were wiped clean with autoclaved gauze before use. The main tool for section preparation in this experiment was a paraffin slicer. Continuous coronal sections were cut with the hippocampal CA1 region as the center, with a thickness of about 5 μm, and attached to clean glass slides treated with polylysine. The prepared sections were dried in an oven at 70°C for 2 - 3 h and then transferred to a 50°C oven for transition. Finally, the baked sections were wrapped with tin foil and then stored in a 4°C refrigerator, ready for subsequent histopathological staining.

[0068] 1. Hematoxylin-eosin staining (HE staining)

[0069] Before HE staining, dewax the pre-prepared tissue sections. The specific operation process is as follows: Immerse the prepared tissue sections in xylene. The first time for 20 minutes, then change the xylene solution and continue to immerse the tissue sections for the second time for 10 minutes to wash away the paraffin. Take out the dewaxed tissue sections and immerse them in 100% ethanol for the first time for 5 minutes, then quickly immerse them in 100% ethanol for the second time for 15 minutes. At 5-minute intervals, starting from 95% ethanol, immerse them in gradient ethanol in the following order (80% → 70% → 50% ethanol) to wash away the xylene on the sections. After the above procedure, rinse with distilled water for 5 minutes. Put the sections into hematoxylin solution for staining for 20 minutes, and wash away the floating color with distilled water. For parts that should not be stained, such as cytoplasm, use 1% hydrochloric acid ethanol to differentiate and wash for a few seconds. Then rinse with distilled water for more than half an hour. Counterstain with 0.5% - 1% eosin solution for 10 minutes to stain the cytoplasm red. Put the counterstained sections into 100% ethanol for 4 - 5 minutes for thorough dehydration, then immerse them in 95% ethanol I and 95% ethanol II for 1 minute each to dehydrate and differentiate the eosin color at the same time. Then put the stained tissue sections into xylene solution for 1 minute for the first transparency treatment, and then put them into xylene solution for 5 minutes for the second thorough transparency treatment. Add 1 - 2 drops of neutral gum on the treated tissue sections, cover with a cover slip for sealing, complete the preparation of tissue sections, and prepare for microscopic observation and photography. In observing various lesions in the brain, including neuronal degeneration, loss, and glial cell hyperplasia, etc., hematoxylin-eosin staining can not only better show the advantages of tissue structure, but also vividly display the pathological morphology of normal and diseased tissues, and count hippocampal neurons and glial cells through the analysis of stained pictures. Apoptotic cells can be observed under an optical microscope and are seen to be scattered individually in the tissue. Phenomena such as dense condensation and fragmentation of nuclear chromatin can be seen. The normal cell nucleus is blue-black and the cytoplasm is light red. Observe with a 10×40 magnification field of view. By the grid method, place the center of the grid in the central area of the hippocampal granular layer, and continuously count the glial cells and pyramidal cells in five grids. Three sections are made for each model mouse, and only the average of the results of the three sections can be used.

[0070] 2. Congo red staining

[0071] Before Congo red staining, dewax the pre-prepared tissue sections. The specific operation method is as follows: First step, soak in xylene solution at 50°C twice with a time interval of 25 minutes for dewaxing; Second step, soak the sections successively in 100%, 95%, 90%, 80% and 75% alcohol at 5-minute intervals to wash off the xylene on the tissue sections; Third step, first rinse the sections with distilled water for 5 minutes, and then immerse them in hematoxylin staining solution for 2 minutes. Differentiate by soaking in 0.5% hydrochloric acid alcohol for a few minutes, without acidification treatment, and wash with distilled water for 5 - 10 minutes; Fourth step, prepare the alkaline working solution by adding 1 mL of 1% sodium hydroxide solution to 100 mL of 80% alcohol saturated sodium chloride solution. The alkaline working solution needs to be prepared freshly before use. Immerse the sections in the prepared alkaline working solution for 10 to 20 minutes without washing; Fifth step, first dissolve 1 g of Congo red in 100 mL of 80% alcohol saturated sodium chloride solution to prepare the Congo red stock solution, and then add 50 mL of the Congo red stock solution to 0.5 mL of 1% sodium hydroxide solution to prepare the Congo red working solution. Immerse the tissue sections in the Congo red working solution for 10 to 20 minutes without excessive washing. Blot the excess Congo red working solution on the tissue sections with absorbent paper; Sixth step, perform gradient dehydration treatment on the stained tissue sections with 50%, 70%, 80%, 90%, 95% and 100% ethanol at 15-second intervals; Seventh step, place the stained tissue sections in xylene solution for 10 minutes for the first transparency treatment, and then place them in the replaced xylene solution for 10 minutes for the second thorough transparency treatment. These stained sections are used to examine the deposition of β-amyloid in the hippocampal tissue of model mice. Under light microscopy, β-amyloid appears dark pink to red, the cell nucleus appears blue, and the cytoplasm is not stained.

[0072] 3. Immunohistochemical staining method

[0073] After the behavioral detection experiment, the experimental animals need to be anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution (10 mg / ml). After placing the operating table, cut the abdominal skin and muscle from under the xiphoid process of the sternum, cut open the diaphragm, fix the ribs cut from both sides of the chest with hemostatic forceps and turn them up to fully expose the heart. Insert a puncture needle into the ascending aorta, fix the needle and cut open the right auricle at the same time. Then, in the first step, quickly inject 50 ml of ice-cold normal saline with a syringe until no bloody liquid flows out of the right auricle and the liver turns yellowish-brown. In the second step, perfuse an appropriate amount of a mixed solution of 4% paraformaldehyde and 2.5% glutaraldehyde until the limbs become rigid. Then decapitate, obtain the complete brain and place it in a paraformaldehyde solution containing 20% sucrose, and fix it at 4°C for 48 h.

[0074] Before staining, dewax the pre-prepared tissue sections. The specific operation process is as follows: Place the specimens dried in the oven in the corresponding solutions in the following sequence and time for treatment (Xylene I for 5 min → Xylene II for 10 min → absolute ethanol → 95% ethanol I → 95% ethanol II → 80% ethanol for 5 s each → wash with water); Place in 3% H2O2 solution at room temperature for 10 min, wash with distilled water 3 times for 5 min each, and then wash with PBS 3 times for 5 min each to eliminate endogenous peroxidase; Microwave repair (thawing temperature from 92°C to 98°C) for 10 min, cool at room temperature and then wash with distilled water 3 times for 5 min each, then wash with PBS 3 times for 5 min each. Finally, after antigen repair with 0.01M citrate antigen repair, add the primary antibody, and then incubate overnight in a 4°C refrigerator; The next day, add the universal immunoglobulin G antibody polymer (secondary antibody) dropwise, incubate in a 37°C incubator for 25 min, wash with PBS 3 times for 5 min each, and then develop color with freshly prepared DAB chromogenic solution (the color development time is controlled within 3 - 5 min); Counterstain with Hematoxylin for 5 - 10 min, wash with distilled water, quickly dip in hydrochloric acid ethanol for differentiation, and then place in warm water for 1 min to complete blue return; Soak successively in 80% ethanol → 95% ethanol I → 95% ethanol II → absolute ethanol for 5 s each for dehydration; Then soak successively in Xylene I and Xylene II for 5 s each for clearing, and select a well-ventilated area in the laboratory to add 1 - 2 drops of neutral gum on the section, cover with a coverslip for sealing, complete the preparation of the tissue section, and prepare for microscopic observation and color pictures, and use the MoticMed6.0 digital medical image analysis software system to calculate the average density and average number of positive cells in the pictures.

[0075] Results:

[0076] 1. The accumulation of Aβ in the mouse brain tissue detected by Aβ42 immunohistochemistry is as Figure 9 shown. It can be seen from the results that the accumulation of Aβ42 in different brain regions of ApoE4(+ / +) / APP / PS1 transgenic mice is different. Compared with wild-type mice, the accumulation in the cerebral cortex is significantly increased, but slightly lower than that in APP / PS1 and ApoE4(+ / +) mice.

[0077] 2. The detection of amyloid deposition in the mouse brain tissue by Congo red staining is as Figure 10 shown. The results show that obvious amyloid deposition appears in ApoE4(+ / +) / APP / PS1 transgenic mice compared with wild-type, APP / PS1, and E4(+ / +) mice, and it exists in all three brain regions, with the largest number in the cortex.

[0078] 3. The detection of cell morphology in the mouse brain tissue by HE staining is as Figure 11As shown. It can be seen from the results that compared with the other three genotypes of mice, the number of cells in the three brain regions of ApoE4(+ / +) / APP / PS1 transgenic mice is significantly reduced, the cell arrangement is not tight, and pathological phenomena such as karyopyknosis and cell degeneration occur.

[0079] Example 5: Expression characteristics of molecular markers in the brain tissue of ApoE4(+ / +) / APP / PS1 transgenic mice

[0080] Aβ protein and tau protein are classical molecular evaluation indicators for Alzheimer's disease brain tissue. APP protein is the precursor of Aβ. Hyperphosphorylation of tau protein will lose its function of binding to microtubules and maintaining the microtubule structure. And hyperphosphorylated tau protein will aggregate and form the pathological phenomenon of neurofibrillary tangles in cells. The expression levels of total tau protein, p-tau and APP protein in the brain tissue of ApoE4(+ / +) / APP / PS1 triple transgenic mice were detected by Western blot method.

[0081] According to the method used in the literature, take 50 μg of protein, first place the sample in a water bath at boiling for 5 min, then in an ice bath for 5 min, and then centrifuge in a low-temperature centrifuge (4 °C) for 5 min at a speed of 12,000 rpm. At the same time, calculate the total volume of the gel required for this experiment, prepare the separating gel solution, and prepare for loading; after accurately installing the electrophoresis device, prepare a certain amount of electrophoresis buffer. After adding an appropriate amount of electrophoresis buffer to the electrophoresis tank, take the supernatant of the pre-treated protein sample for loading. The loading amount of the sample is 20 μL, and the loading amount of the protein Marker is 10 μL. The voltages of the stacking gel and the separating gel are set to 80 V and 120 V respectively; after the electrophoresis process is completed, cut a PVDF membrane with the same size as the gel, and place it in methanol for about 30 s for pretreatment; after the pretreatment is completed, first make a transfer membrane sandwich, and then transfer the membrane at a constant voltage of 80 V at 4 °C for about 2 h; after the transfer, wet the PVDF membrane from bottom to top with TBST, then transfer it to a petri dish with blocking solution and place it on a shaker, and shake it at room temperature for 2 h; block it with 5% skim milk powder [5% (w / v) skim milk powder / TBST buffer] at room temperature for 2 h; after the blocking is completed, wash the PVDF membrane 5 times with TBST on a shaker at room temperature, 10 min each time; dilute the primary antibody to the required concentration with TBST solution, and incubate it with shaking at 4 °C overnight; the next day, wash the PVDF membrane 5 times with TBST on a shaker at room temperature, 10 min each time; finally, dilute the secondary antibody to the required concentration with TBST solution, incubate it with shaking at 37 °C for 1 h, then place it at room temperature for 2 h, wash it 3 times with TBST on a shaker, 10 min each time, then develop color, and finally use a gel imager to collect images and analyze the gel images with special analysis software.

[0082] The results of detecting the total tau protein content in the hippocampal tissue of mouse brains by Western blot are as Figure 12 shown. The results of detecting the p-tau protein content in the hippocampal tissue of mouse brains by Western blot are as Figure 13 shown. The results of detecting the APP protein content in the hippocampal tissue of mouse brains by Western blot are as Figure 14 shown.

[0083] The results show that the expression of total Tau protein in the brain tissue of ApoE4(+ / +) / APP / PS1 transgenic mice is significantly higher than that of the other three types of mice; the levels of p-Tau and APP are significantly higher than those of E4(+ / +). This indicates that ApoE4(+ / +) / APP / PS1 transgenic mice have the pathological characteristics of tau protein deposition in Alzheimer's disease and there is an abnormal increase in APP.

[0084] For some implementations, if they are not the key content of the present invention and are well-known to those of ordinary skill in the art, due to space limitations, they are not described in detail in the specification drawings or the text. In this case, reference can be made to the relevant prior art for understanding. Moreover, the purpose of providing the above embodiments is only to make the present invention meet legal requirements, and the present invention can be implemented in many different forms and should not be construed as limited to the embodiments described herein.

[0085] Similarly, it should be understood that, in order to streamline the present invention, in the above description of the exemplary embodiments of the present invention, the various features of the present invention are sometimes grouped together into a single embodiment, figure, or description thereof. However, the method of the invention should not be construed as reflecting the intention that the claimed invention requires more features than those expressly recited in each claim. More precisely, as reflected by the claims, each aspect of the invention lies in less than all the features of the preceding single embodiment. And, the embodiments can be combined with each other or with other embodiments based on design and reliability considerations, that is, the technical features in different embodiments can be freely combined to form more embodiments. Therefore, the claims following the specific embodiments are hereby expressly incorporated into the specific embodiments, where each claim itself serves as a separate embodiment of the present invention.

[0086] In the above specific embodiments, the purpose, technical means, and beneficial effects of the present invention are described in detail. It should be understood that the purpose of the detailed description is for those skilled in the art to understand the present invention more clearly and is not used to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for constructing an Alzheimer's disease mouse model, characterized in that: include: ApoE4(+ / +) transgenic mice were hybridized with APP / PSEN1 double transgenic mice, and the obtained offspring were identified; the first generation offspring with the genotype of ApoE4(+ / -) / APP / PSEN were hybridized, and the obtained second generation offspring were identified to obtain a homozygous positive ApoE4(+ / +) / APP / PSEN1 Alzheimer's disease mouse model.

2. The method according to claim 1, characterized in that: The obtained second-generation mice were identified, including the ApoE4(+ / +) / APP / PSEN1 mice according to the molecular, pathological, and behavioral characteristics of Alzheimer's disease.

3. The method according to claim 1, characterized in that The method for preparing the ApoE4(+ / +) transgenic mouse comprises: The human ApoE4 transgenic fragment was injected into mouse fertilized eggs by microinjection technology; The surviving fertilized eggs were transplanted into pseudo-pregnant mother mice to obtain the ApoE4 (+ / +) transgenic mice.

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