Animal model construction method based on APOE gene knockout and application thereof

Abstract

The invention discloses an animal model construction method based on APOE gene knockout and application of the animal model construction method, and belongs to the technical field of biomedical experimental models.6-8-week male ApoE-KO mice are randomly divided into three groups, each group comprises 10 mice, and the first group is fed with common diet and serves as a baseline model to simulate genetic susceptibility; the second group of ApoE-KO mice are fed with high fat diet (HFD), and double effects of inheritance and environment are simulated; the third group of ApoE-KO mice are fed with high fat diet HFD, H1152 is injected to verify the inhibition effect of the Rho kinase inhibitor after the third group of ApoE-KO mice are fed with the high fat diet HFDD for 4 weeks, the medicine intervention effect is evaluated, the injection mode is intraperitoneal injection, and the medicine intervention effect is evaluated. The grouping mode can be expanded to multi-factor mechanism research of other metabolic diseases such as diabetes, non-alcoholic fatty liver and neurodegenerative diseases such as Alzheimer's disease, and a model basis is provided for precise medical pharmacy.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical experimental models, and specifically relates to a method for constructing an animal model based on APOE gene knockout and its application. Background Art

[0002] In traditional atherosclerosis research, single-factor models (such as simple ApoE-KO mice) or two-factor models (such as ApoE-KO + high-fat diet) are often used, but they cannot comprehensively simulate the multi-factor synergistic mechanism of genes, environment, drug intervention, etc. in the disease process.

[0003] There is a synergistic effect between lipid metabolism disorders caused by gene defects (such as ApoE-KO) and metabolic stress induced by high-fat diet (HFD), but existing models are difficult to quantify the independent contributions of the two to vascular inflammation and plaque formation. Further, when drug intervention is introduced, its effect may be masked by the superimposed effects of genes or diet. It is impossible to distinguish the independent and interactive effects of gene defects, diet induction, and drug intervention on disease phenotypes.

[0004] The existing drug efficacy evaluation system generally has problems such as incomplete grouping logic and insufficient control group settings, resulting in limited reliability of target validation. Most studies do not set up a "basic pathology group" (such as ApoE-KO), an "environmental stress group" (such as ApoE-KO + HFD), or a "drug intervention group" (such as ApoE-KO + HFD + H1152), so it is impossible to clarify whether the drug effect depends on specific environmental factors (such as metabolic stress induced by HFD). There is a lack of a standardized grouping mode to systematically evaluate the efficacy of potential therapeutic targets (such as the Rho kinase inhibitor H1152). Summary of the Invention

[0005] Technical Problems to be Solved

[0006] Aiming at the deficiencies of the existing technology, the present application solves the technical problems that it is currently impossible to comprehensively simulate the multi-factor synergistic mechanism of genes, environment, drug intervention, etc. in the disease process, it is impossible to distinguish the independent and interactive effects of gene defects, diet induction, and drug intervention on disease phenotypes, and there is a lack of a standardized grouping mode to systematically evaluate the efficacy of potential therapeutic targets. The present application provides a method for constructing an animal model based on APOE gene knockout and its application, which is used to systematically study the pathogenesis of atherosclerosis (AS) and the effect of drug intervention.

[0007] Technical Solutions

[0008] To achieve the above object, the present application is realized through the following technical solutions:

[0009] A method for constructing an animal model based on APOE gene knockout specifically includes the following steps:

[0010] Step 1: Construct the experimental groups with simple gene defect ApoE-KO: Male ApoE-KO mice at 6-8 weeks old were randomly divided into 3 groups, with 10 mice in each group. The first group was fed a normal diet as the baseline model to simulate genetic susceptibility.

[0011] Step 2: Construct the experimental groups with gene defect combined with high-fat diet-induced ApoE-KO+HFD: The second group of ApoE-KO mice was fed a high-fat diet HFD to simulate the dual effects of genetics and environment.

[0012] Step 3: Construct the experimental groups with gene defect + high-fat diet + H1152 intervention ApoE-KO+HFD+H1152: The third group

[0013] ApoE-KO mice were fed a high-fat diet HFD. After 4 weeks of feeding the high-fat diet HFD, H1152 was injected to verify the inhibitory effect of the Rho kinase inhibitor. The ApoE-KO mice with H1152 combined with a high-fat diet were used to evaluate the drug intervention effect. The injection method was intraperitoneal injection.

[0014] Furthermore, the high-fat diet HFD specifically consisted of 40% kcal% fat, 40% kcal% carbohydrate, and 20% kcal% protein for 12 weeks.

[0015] Furthermore, the dosing dose of H1152 was 10 mg / kg / d, by intraperitoneal injection, for 8 weeks, once every other day.

[0016] Furthermore, the specific method for verifying the inhibitory effect of the Rho kinase inhibitor was to observe the changes in plaque area, blood cells, and plasma parameters.

[0017] Furthermore, the plaque area was the area of oil red-stained plaques; the blood cells were neutrophils, lymphocytes, monocytes, eosinophils, and basophils; the changes in plasma parameters were the changes in total cholesterol TC, triglyceride TG, high-density lipoprotein cholesterol HDL-C, low-density lipoprotein cholesterol LDL-C, and blood glucose parameters.

[0018] This application also discloses the use of the animal model based on APOE gene knockout obtained by any of the above construction methods in evaluating Rho kinase inhibitors.

[0019] Use of an animal model based on APOE gene knockout obtained by any of the above construction methods in evaluating the efficacy of cardiovascular drugs. Principle explanation: Based on the ApoE knockout animal model, this application constructs a triple control experiment system of "simple gene defect", "gene defect superimposed with high-fat diet induction", and "gene defect + high-fat diet + H1152 intervention" to develop an atherosclerotic targeted therapeutic drug for the Rho kinase signaling pathway; proposes a multi-level grouping mode, constructs a dynamic research system by gradually superimposing gene defects (ApoE-KO), environmental factors (high-fat diet), and drug intervention (H1152), and accurately analyzes the development and intervention mechanism of atherosclerosis; by comparing the biological differences in vascular endothelial function, plasma parameters, blood cell content changes, and arterial plaque formation under different intervention conditions, reveals the synergistic mechanism of H1152 (a specific inhibitor of Rho kinase) in regulating reverse cholesterol transport, inhibiting macrophage foam cell formation, and maintaining vascular homeostasis; this application focuses on protecting the use of this compound in improving dyslipidemia-related arteriosclerosis, especially by double-blocking the pathological processes induced by gene defects and diet, providing an innovative therapeutic drug strategy for patients with familial hypercholesterolemia.

[0020] Beneficial effects:

[0021] This application provides a method for constructing an animal model based on APOE gene knockout. Compared with the prior art, it has the following beneficial effects:

[0022] 1. This grouping mode can be extended to the multi-factor mechanism research of other metabolic diseases such as diabetes, non-alcoholic fatty liver, and neurodegenerative diseases such as Alzheimer's disease, providing a model basis for precision medical therapeutic drugs.

[0023] 2. After HFD high-fat diet treatment, plasma TC, TG, LDL-C, and Glucose were significantly increased, proving that HFD accelerates AS; H1152 treatment decreased TC, TG, LDL-C, and Glucose and significantly reduced the plaque area of the thoracic and abdominal aorta, indicating that H1152 alleviates AS by inhibiting the Rho / ROCK pathway. Brief description of the drawings

[0024] Figure 1 It is a schematic diagram of the inhibitory effect of H1152 on the Rho / ROCK signaling pathway in this application, where the left figure is the WB detection effect diagram and the right figure is the WB detection quantitative diagram;

[0025] Figure 2 It is a comparison diagram of the plasma TC total cholesterol content of three treatment groups in this application;

[0026] Figure 3 It is a comparison diagram of the plasma TG triglyceride content of three treatment groups in this application;

[0027] Figure 4 This is the comparison chart of plasma LDL-C (low-density lipoprotein cholesterol) content of three treatment groups in this application;

[0028] Figure 5 This is the comparison chart of plasma HDL-C (high-density lipoprotein cholesterol) content of three treatment groups in this application;

[0029] Figure 6 This is the comparison chart of plasma Glucose content of three treatment groups in this application;

[0030] Figure 7 This is the percentage chart of the content of Neutrophiles, Lymphocyte, Monocyte, Eosinophils, and Basophils after H1152 treatment in this application;

[0031] Figure 8 This is the comparison chart of abdominal and thoracic aortic plaques after H1152 treatment in this application. The left figure is the schematic comparison of abdominal and thoracic aortic plaques between the H1152 treatment group (ApoE knockout mice with H1152 superimposed on high-fat diet) and the control group (ApoE knockout mice with simple high-fat diet), and the right figure is the quantitative chart of the plaque areas of the two groups. Detailed implementation manners

[0032] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and the equivalent forms of changes or modifications also fall within the scope defined by the claims of this application.

[0033] The ApoE-KO mice are 6-8 weeks old; sex: male; strain: C57BL6, and are purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd.

[0034] Example 1:

[0035] A method for constructing an animal model based on APOE gene knockout specifically includes the following steps:

[0036] The first step is to construct a simple gene defect ApoE-KO experimental grouping: 6-8 week-old male ApoE-KO mice are randomly divided into 3 groups, with 10 mice in each group. The first group is fed a normal diet as a baseline model to simulate genetic susceptibility;

[0037] Step 2: Establish the experimental groups of ApoE-KO+HFD induced by gene deficiency and high-fat diet: In the second group, ApoE-KO mice were fed a high-fat diet (HFD) to simulate the dual effects of genetics and environment;

[0038] Step 3: Establish the experimental groups of ApoE-KO+HFD+H1152 intervened by gene deficiency, high-fat diet and H1152: In the third group

[0039] ApoE-KO mice were fed a high-fat diet (HFD). After 4 weeks of feeding the high-fat diet (HFD), H1152 was injected to verify the inhibitory effect of the Rho kinase inhibitor. ApoE-KO mice with H1152 superimposed on the high-fat diet were used to evaluate the drug intervention effect. The injection method was intraperitoneal injection. During the construction process of the three groups of animal models, pay attention to the dental conditions of rodents at any time to prevent long teeth caused by long-term consumption of high-fat food.

[0040] The high-fat diet (HFD) specifically consisted of 40% kcal% fat, 40% kcal% carbohydrate, and 20% kcal% protein and was fed for 12 weeks.

[0041] The administration dose of H1152 was 10 mg / kg / d, by intraperitoneal injection, for 8 weeks, once every other day.

[0042] The specific method for verifying the inhibitory effect of the Rho kinase inhibitor was to observe the changes in plaque area, blood cells and plasma parameters; the plaque area was the area of the oil red-stained plaque; the blood cells were neutrophils, lymphocytes, monocytes, eosinophils and basophils; the changes in plasma parameters were the changes in total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and blood glucose parameters.

[0043] Example 2:

[0044] For the method for constructing an animal model based on APOE gene knockout in Example 1, the specific steps for verifying the inhibitory effect of the Rho kinase inhibitor are as follows:

[0045] First step: After weighing the body weight of the mice, the mice were euthanized; blood was collected from the hearts of the mice to detect changes in blood cell composition, and the plasma after centrifugation was used for the detection of plasma biochemical indexes of the mice. The thoracic and abdominal aortas of the mice were collected for Western blot and oil red staining to detect lipid deposition. The aortic arch and heart were embedded and frozen sectioned to evaluate the lipid deposition and cell component changes at the plaque-prone sites;

[0046] Step 2, Analysis of mouse blood cell components: Collect the whole blood of mice and immediately detect it on an automatic hematology analyzer to read the total number of white blood cells (WBC), and the percentages of neutrophils, lymphocytes, monocytes, eosinophils, and basophils, so as to analyze the blood cell components of the control and experimental group mice;

[0047] Step 3, Detection of mouse plasma components: Centrifuge at 6000 rpm for 15 min at 4 °C to collect the mouse plasma, and then use a kit to detect the differences in the contents of total cholesterol (T-CHO), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and glucose in the plasma of the two groups, as follows:

[0048] (1) Operation table for T-CHO determination

[0049] Sample addition system for 96-well plate:

[0050]

[0051]

[0052] After mixing, incubate at 37 °C for 10 min and measure the absorbance at a wavelength of 510 nm.

[0053]

[0054] The concentration of the calibrator is: 5.17 mM;

[0055] (2) Operation table for TG determination

[0056] Sample addition system for 96-well plate:

[0057] Blank hole (μl) Calibration hole (μl) Sample hole (μl) <![CDATA[ddH2O]]> 2.5 - - Calibrator - 2.5 - Sample - - 2.5 Working solution 250 250 250

[0058] After mixing, incubate at 37 °C for 10 min and measure the absorbance of each well at a wavelength of 500 nm.

[0059]

[0060] The concentration of the calibrator is: 2.26 mM;

[0061] (3) Operation table for HDL-C determination

[0062] Sample addition system for 96-well plate:

[0063]

[0064]

[0065] The calibrator concentration is: 1.8 mM;

[0066] (4), LDL-C determination operation table

[0067] 96-well plate sample addition system:

[0068]

[0069]

[0070] Or directly calculated by the formula LDL-C = TC - HDLC - TG / 2.2.

[0071] (5), Glucose determination operation table

[0072] 96-well plate sample addition system:

[0073]

[0074] After mixing, incubate at 37 °C for 10 min, wavelength 505 nm, and measure the absorbance value of each well with an enzyme-labeled instrument.

[0075]

[0076] Glucose (mg / dL) = mM × 18

[0077] The calibrator concentration is: 5.55 mM;

[0078] Fourth step, Oil Red O staining of the thoracic and abdominal aorta:

[0079] Step a, Preparation before staining: Weigh 3 g of Oil Red O dye powder into 600 mL of propylene glycol solution, heat and dissolve it in a 95 °C metal bath with stirring, and filter it with a double-circle qualitative filter paper while heating. Let the filtered Oil Red O solution stand at room temperature for one day, and filter it with a 0.45 μm filter before use. Note to store it away from light;

[0080] Step b, staining step: The collected thoracic and abdominal aorta is stored and fixed in a centrifuge tube containing 10% neutral formalin. Before staining, the adipose tissue and connective tissue attached to the surface need to be carefully removed with ophthalmic scissors, taking care not to damage the integrity of the aorta. Then it is placed in PBS and gently shaken overnight on a shaker at 4°C. The next day, it is taken out of the PBS, slightly dried and then placed in propylene glycol for 2 minutes, and then placed in the pre-prepared oil red staining solution for 3 hours. After the staining is completed, it is taken out and passed through 85% propylene glycol three times. At this time, the peripheral staining of the blood vessel is completed. The blood vessel is longitudinally dissected under a stereomicroscope and carefully fixed with a fine needle, taking care not to damage the plaque structure inside the blood vessel during this process. The fixed blood vessel is repeated the above staining steps, 2 minutes in propylene glycol, 3 hours of oil red staining, 3 times through 85% propylene glycol, and then 3 times in PBS, and placed on a shaker at 4°C and shaken overnight. Take pictures under a stereomicroscope and perform quantitative analysis with Image-Pro Plus software.

[0081] Result analysis:

[0082] After HFD high-fat diet treatment, plasma TC, TG, LDL-C, and Glucose were significantly increased, proving that HFD accelerates AS; H1152 treatment decreased TC, TG, LDL-C, and Glucose and significantly reduced the plaque area of the thoracic and abdominal aorta, indicating that H1152 relieves AS by inhibiting the Rho / ROCK pathway.

[0083] The above is an exemplary description of the present invention. It should be noted that without departing from the core of the present invention, any simple deformation, modification or equivalent substitution that can be made by those skilled in the art without creative labor falls within the protection scope of the present invention.

Claims

1. A method for constructing an animal model based on APOE gene knockout, characterized in that, Specifically, the following steps are included: In the first step, construct a simple gene-deficient ApoE-KO experimental grouping: male ApoE-KO mice at 6-8 weeks old are randomly divided into 3 groups, with 10 mice in each group. The first group is fed a normal diet as a baseline model to simulate genetic susceptibility; In the second step, construct a gene-deficient plus high-fat diet-induced ApoE-KO+HFD experimental grouping: the second group of ApoE-KO mice is fed a high-fat diet HFD to simulate the dual effects of genetics and environment; In the third step, construct a gene-deficient + high-fat diet + H1152 intervention ApoE-KO+HFD+H1152 experimental grouping: the third group of ApoE-KO mice is fed a high-fat diet HFD. After 4 weeks of feeding the high-fat diet HFD, H1152 is injected to verify the inhibitory effect of the Rho kinase inhibitor. The ApoE-KO mice with H1152 plus high-fat diet are used to evaluate the drug intervention effect. The injection method is intraperitoneal injection.

2. The method for constructing an animal model based on APOE gene knockout according to claim 1, wherein: High-fat diet HFD Specifically, it is fed with 40% kcal% fat, 40% kcal% carbohydrate, and 20% kcal% protein for 12 weeks.

3. The method for constructing an animal model based on APOE gene knockout according to claim 1, wherein: H1152 administration The drug dosage is 10 mg / kg / d, intraperitoneal injection, for 8 weeks, once every other day.

4. The method for constructing an animal model based on APOE gene knockout according to claim 1, characterized in that: The specific method for verifying the inhibitory effect of the Rho kinase inhibitor is to observe the changes in plaque area, blood cells, and plasma parameters.

5. The method for constructing an animal model based on APOE gene knockout according to claim 4, wherein: The plaque area is the area of the oil red-stained plaque; the blood cells are neutrophils, lymphocytes, monocytes, eosinophils, and basophils; the changes in plasma parameters are the changes in total cholesterol TC, triglyceride TG, high-density lipoprotein cholesterol HDL-C, low-density lipoprotein cholesterol LDL-C, and blood glucose parameters.

6. Use of an animal model based on APOE gene knockout obtained by the construction method according to any one of claims 1-5 in evaluating a Rho kinase inhibitor.

7. Use of an animal model based on APOE gene knockout obtained by the construction method according to any one of claims 1-5 in evaluating the efficacy of cardiovascular drugs.

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