Ob / ob obese mouse model with atherosclerosis as well as construction method and application of ob / ob obese mouse model

Abstract

The invention relates to the technical field of animal model construction, in particular to an ob / ob obese mouse model with atherosclerosis as well as a construction method and application of the ob / ob obese mouse model. The invention provides a construction method of an ob / ob obese mouse model with atherosclerosis, which comprises the following steps: providing an ob / ob mouse, and injecting AAV8-PCSK9 virus into the ob / ob mouse through tail intravenous injection; and feeding with high-fat feed to obtain the ob / ob obese mouse model with atherosclerosis. The construction method is simple, does not need to use large-scale instruments and equipment, and is short in time and easy to operate.

Description

Technical Field

[0001] The present application belongs to the technical field of animal model construction, and in particular relates to an ob / ob obese mouse model of atherosclerosis and a construction method and application thereof. Background Art

[0002] Atherosclerosis is a multifactorial disease involving chronic inflammation and metabolic disorders in multiple tissue types. The incidence of coronary heart disease increases dramatically in patients with metabolic syndrome. There is now a large body of basic and clinical evidence showing a correlation between metabolically impaired adipose tissue and the atherosclerotic vasculature. When obesity occurs, adipocyte differentiation is impaired, adiponectin production is reduced, and the secretion of proinflammatory cytokines such as tumor necrosis factor, interleukin-6, and monocyte chemoattractant protein increases, thereby promoting the development of atherosclerosis.

[0003] Animal models of atherosclerosis play a very important role in exploring the pathogenesis and pathological process of the disease and guiding clinical treatment. Animals used to establish atherosclerosis models include rats, mice, miniature pigs, rabbits and primates, among which mice are the most widely used in basic research. Commonly used methods for establishing mouse atherosclerosis models include gene regulation, high-fat induction and mechanical injury (arterial clamping, balloon injury), etc. Among them, apolipoprotein E (ApoE) and low-density lipoprotein receptor (LDLR) gene knockout mice are the most classic transgenic mouse models. After combined high-fat diet feeding, they promote the formation of atherosclerotic plaques in mice by inducing severe hyperlipidemia. However, there is currently no mouse model suitable for studying obesity-related atherosclerosis.

[0004] Therefore, establishing an obesity-related mouse atherosclerosis model is crucial for basic research on atherosclerosis. Providing a suitable animal model for studying the relationship between obesity and atherosclerosis will help researchers further clarify the regulatory mechanism of obesity / fat on atherosclerosis, thereby guiding clinical treatment. Summary of the invention

[0005] The purpose of the present application is to provide an ob / ob obese mouse model of atherosclerosis and a construction method and application thereof, aiming to solve the problem that there is no mouse model of obesity-related atherosclerosis in the prior art.

[0006] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0007] In a first aspect, the present application provides a method for constructing an ob / ob obese mouse model of atherosclerosis, comprising the following steps: providing ob / ob mice, injecting the AAV8-PCSK9 virus into the ob / ob mice via the tail vein; and then feeding the ob / ob mice with a high-fat diet to obtain an ob / ob obese mouse model of atherosclerosis.

[0008] In some embodiments, the body weight of the ob / ob mouse is 40-50 g / mouse.

[0009] In some embodiments, the ob / ob mouse is an 8 week old male ob / ob mouse.

[0010] In some embodiments, a method for breeding 8-week-old male ob / ob mice comprises: raising the male ob / ob mice under pathogen-free conditions of 19-23°C, 40%-60% relative humidity, repeatedly carrying out a cycle of 12 hours of light cultivation and 12 hours of dark cultivation until they are 8 weeks old, thereby obtaining 8-week-old male ob / ob mice.

[0011] In some embodiments, the injection volume of AAV8-PCSK9 virus is 1×10^11 vg / mouse.

[0012] In some embodiments, the high-fat diet is selected from Research Diets' feed with catalog number D12109C.

[0013] In a second aspect, the present application provides an ob / ob obese mouse model of atherosclerosis, wherein the ob / ob obese mouse model of atherosclerosis is constructed by the above-mentioned method for constructing an ob / ob obese mouse model of atherosclerosis.

[0014] In a third aspect, the present application provides an application of the method for constructing the above-mentioned ob / ob obese mouse model of atherosclerosis in studying the relationship between obesity and atherosclerosis.

[0015] In some embodiments, the ob / ob obese mouse model of atherosclerosis constructed by the construction method is used to develop the regulatory mechanism of obesity on atherosclerosis.

[0016] In some embodiments, the ob / ob obese mouse model of atherosclerosis constructed by the construction method is used to develop therapeutic targets and therapeutic drugs for obesity-related atherosclerosis.

[0017] The first aspect of the present application provides a method for constructing an ob / ob obese mouse model of atherosclerosis. The method is to use ob / ob obese mice as the subjects, inject AAV8-PCSK9 virus into the tail vein of the mice, and then feed them a high-fat diet. Since PCSK9 is highly expressed in the liver, it binds to LDLR and promotes its degradation in hepatocytes, thereby blocking the circulation pathway of LDLR on the cell membrane surface, inhibiting the clearance of low-density lipoprotein cholesterol (LDL-C), increasing LDL-C in plasma, causing hyperlipidemia, and leading to the formation of atherosclerosis. Therefore, an obesity-related atherosclerosis mouse model is established. The construction method is simple, does not require the use of large instruments and equipment, is time-saving, and is easy to operate.

[0018] The ob / ob obese mouse model of atherosclerosis provided in the second aspect of the present application is constructed by the above construction method. The obtained animal model is crucial for basic research on atherosclerosis, provides a suitable animal model for studying the relationship between obesity and atherosclerosis, and helps researchers further clarify the regulatory mechanism of obesity / fat on atherosclerosis, thereby guiding clinical treatment.

[0019] The third aspect of the present application provides a method for constructing an ob / ob obese mouse model of atherosclerosis and its application in studying the relationship between obesity and atherosclerosis; the provided model conforms to the current pathogenesis of obesity-related atherosclerosis in humans and can be used to study the regulatory mechanism of obesity-related atherosclerosis and to find relevant targets for early diagnosis and preventive and therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 This is a weight analysis chart of the mouse model provided in Example 1 of the present application.

[0022] Figure 2 This is an analysis diagram of atherosclerotic plaques in the mouse model provided in Example 1 of the present application. DETAILED DESCRIPTION

[0023] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0024] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0025] In this application, "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0026] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0027] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "an" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0028] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass in the embodiment description of the present application can be μg, mg, g, kg and other mass units known in the chemical industry.

[0029] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0030] Leptin is a hormone that regulates appetite. It is the expression product of the ob gene and is mainly secreted by fat cells. It can suppress appetite, increase energy consumption, inhibit fat synthesis and promote fat decomposition. The ob gene is located on chromosome 6 of mice. When its coding region mutates, that is, cytosine C is replaced by thymine T, and CGA becomes the termination codon TGA, the synthesis of arginine at position 105 is terminated prematurely, and a large amount of non-functional leptin is generated, resulting in obesity.

[0031] ob / ob mice refer to a genetic mouse model widely used to study obesity and type 2 diabetes. The ob (obese) gene mutation carried by these mice leads to leptin deficiency and they show severe obesity, insulin resistance and high sugar symptoms, which are very similar to human obesity. They can be used for basic research on the pathophysiology of obesity, hormone levels and drug treatment.

[0032] The first aspect of the present application provides a method for constructing an ob / ob obese mouse model of atherosclerosis, comprising the following steps:

[0033] S01. provide ob / ob mice, and inject AAV8-PCSK9 virus into the ob / ob mice via tail vein;

[0034] S02. Feed the mice with a high-fat diet to obtain an ob / ob obese mouse model with atherosclerosis.

[0035] The method for constructing an ob / ob obese mouse model of atherosclerosis provided in the first aspect of the embodiment of the present application is to use ob / ob obese mice as the subjects, inject AAV8-PCSK9 virus into the tail vein of the mice, and then feed them a high-fat diet. Since PCSK9 is highly expressed in the liver, it binds to LDLR and promotes its degradation in hepatocytes, thereby blocking the circulation pathway of LDLR on the cell membrane surface, inhibiting the clearance of low-density lipoprotein cholesterol (LDL-C), increasing LDL-C in plasma, causing hyperlipidemia, and leading to the formation of atherosclerosis. Therefore, an obesity-related atherosclerosis mouse model is established. The construction method is simple, does not require the use of large instruments and equipment, is time-saving, and is easy to operate.

[0036] In step S01, ob / ob mice are provided, and AAV8-PCSK9 virus is injected into the ob / ob mice via the tail vein.

[0037] In some embodiments, the weight of ob / ob mice is 40-50 g / mouse. In some specific embodiments, the weight of ob / ob mice includes, but is not limited to, 40 g / mouse, 41 g / mouse, 42 g / mouse, 43 g / mouse, 44 g / mouse, 45 g / mouse, 46 g / mouse, 47 g / mouse, 48 g / mouse, 49 g / mouse, 50 g / mouse, and other typical but non-limiting arbitrary point values ​​or interval values ​​between any two point values.

[0038] In some embodiments, the ob / ob mouse is an 8 week old male ob / ob mouse.

[0039] In some embodiments, a method for breeding 8-week-old male ob / ob mice comprises: raising the male ob / ob mice under pathogen-free conditions of 19-23°C, 40%-60% relative humidity, repeatedly carrying out a cycle of 12 hours of light cultivation and 12 hours of dark cultivation until they are 8 weeks old, thereby obtaining 8-week-old male ob / ob mice.

[0040] Furthermore, AAV8-PCSK9 virus was injected into ob / ob mice through the tail vein. Since PCSK9 is highly expressed in the liver, it binds to LDLR and promotes its degradation in hepatocytes, thereby blocking the circulation pathway of LDLR on the cell membrane surface, inhibiting the clearance of low-density lipoprotein cholesterol (LDL-C), increasing LDL-C in plasma, causing hyperlipidemia, and leading to the formation of atherosclerosis. Therefore, an obesity-related atherosclerosis mouse model can be constructed.

[0041] In some embodiments, the injection volume of AAV8-PCSK9 virus is 1×10^11 vg / mouse. If the injection volume of AAV8-PCSK9 virus is too low, the modeling will fail and atherosclerosis will not be induced; if the injection volume is too high, the mice may die due to severe hyperlipidemia, inflammatory response and atherosclerosis.

[0042] In step S02, the mice are fed with a high-fat diet to obtain an ob / ob obese mouse model of atherosclerosis.

[0043] In some embodiments, the high-fat diet is selected from Research Diets' feed with catalog number D12109C.

[0044] In some embodiments, during high-fat feeding, fresh high-fat feed is replaced regularly every day, and mice have free access to food and water.

[0045] In some embodiments, the high-fat diet is fed for 8 weeks to obtain the ob / ob obese mouse model of atherosclerosis.

[0046] A second aspect of an embodiment of the present application provides an ob / ob obese mouse model of atherosclerosis. The ob / ob obese mouse model of atherosclerosis is constructed by the above-mentioned method for constructing an ob / ob obese mouse model of atherosclerosis.

[0047] The ob / ob obese mouse model of atherosclerosis provided in the second aspect of the embodiment of the present application is constructed by the above construction method. The obtained animal model is crucial for basic research on atherosclerosis, provides a suitable animal model for studying the relationship between obesity and atherosclerosis, and helps researchers to further clarify the regulatory mechanism of obesity / fat on atherosclerosis, thereby guiding clinical treatment.

[0048] The third aspect of the embodiments of the present application provides the application of the method for constructing the above-mentioned ob / ob obese mouse model of atherosclerosis in studying the relationship between obesity and atherosclerosis.

[0049] The third aspect of the embodiment of the present application provides a method for constructing an ob / ob obese mouse model of atherosclerosis and its application in studying the relationship between obesity and atherosclerosis; the provided model conforms to the current pathogenesis of obesity-related atherosclerosis in humans and can be used to study the regulatory mechanism of obesity-related atherosclerosis and to find relevant targets for early diagnosis and preventive and therapeutic drugs.

[0050] In some embodiments, the ob / ob obese mouse model of atherosclerosis constructed by the construction method is used to develop the regulatory mechanism of obesity on atherosclerosis.

[0051] In some embodiments, the ob / ob obese mouse model of atherosclerosis constructed by the construction method is used to develop therapeutic targets and therapeutic drugs for obesity-related atherosclerosis.

[0052] The following describes the invention in conjunction with specific embodiments.

[0053] Example 1

[0054] The method for constructing the ob / ob obese mouse model of atherosclerosis is

[0055] Male ob / ob mice were raised under pathogen-free conditions of 19-23°C, 40%-60% relative humidity, and 12-hour light and dark cycles until they were 8 weeks old, thereby obtaining 8-week-old male ob / ob mice; ob / ob mice were injected with AAV8-PCSK9 virus via the tail vein, and the injection amount of AAV8-PCSK9 virus was 1×10^11 vg / mouse;

[0056] The mice were then fed with a high-fat feed with the product number D12109C from Research Diets. During the high-fat feeding process, the high-fat feed had to be replaced with fresh high-fat feed on a regular basis every day. The mice were allowed to drink water freely. After 8 weeks of feeding, an ob / ob obese mouse model of atherosclerosis was obtained.

[0057] Performance Testing

[0058] 1. Body weight test. The constructed mouse model was tested for body weight every week.

[0059] 2. Analyze atherosclerotic plaques. The analysis method is: use CO 2 Methods: Mice were killed, and 10 mL of phosphate-balanced saline was used to perfuse the whole body of the mice, followed by perfusion of 4% paraformaldehyde through the left ventricle. The entire aortic branch including the heart was separated, the perivascular fat and other excess tissues were removed, the aorta was longitudinally cut open, and Oil Red O was used for staining. The saturated Oil Red O dye solution and distilled water were diluted 3:2 one day in advance, and allowed to stand overnight at 4°C. The next day, it was filtered with qualitative filter paper to obtain the Oil Red O working solution. The cut aorta was placed in the Oil Red O working solution, incubated and stained at 37°C for 15 minutes, the aorta was removed, immersed in 60% isopropanol for 10 seconds, and then rinsed in pure water twice, 1-2 minutes each time. The stained tissue was fixed and then digitally scanned.

[0060] Results Analysis

[0061] 1. Weight analysis

[0062] Figure 2. Weight gain in the ob / ob obese mouse model of atherosclerosis. Figure 1 As shown: 3 weeks after modeling, the weight of mice decreased slightly, and then gradually increased. In the 7th week, the weight of mice could reach about 58 grams.

[0063] 2. Analyze atherosclerotic plaques.

[0064] Analysis of atherosclerotic plaques Figure 2 As shown (the three mice were all mice after modeling for 8 weeks, and were regarded as parallel experiments), among which, Figure 2 The percentage of atherosclerotic plaques in A to the total area was 10.51%. Figure 2 The percentage of atherosclerotic plaques in B's total area was 5.97%. Figure 2 The percentage of atherosclerotic plaques in the total area of ​​C was 10.60%. After 8 weeks of modeling, it was clearly seen that there were obvious atherosclerotic plaques in the aortic tree of ob / ob mice.

[0065] In summary, the method for constructing an ob / ob obese mouse model of atherosclerosis provided in the embodiments of the present application is to use ob / ob obese mice as the subjects, inject AAV8-PCSK9 virus into the tail vein of the mice, and then feed them a high-fat diet. Since PCSK9 is highly expressed in the liver, it binds to LDLR and promotes its degradation in hepatocytes, thereby blocking the circulation pathway of LDLR on the cell membrane surface, inhibiting the clearance of low-density lipoprotein cholesterol (LDL-C), increasing LDL-C in plasma, causing hyperlipidemia, and leading to the formation of atherosclerosis. Therefore, an obesity-related atherosclerosis mouse model is established. The construction method is simple, does not require the use of large instruments and equipment, is time-saving, and is easy to operate.

[0066] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for constructing an ob / ob obese mouse model of atherosclerosis, characterized in that: The method comprises the following steps: providing ob / ob mice, injecting AAV8-PCSK9 virus into the ob / ob mice via tail vein; and then feeding the ob / ob mice with high-fat diet to obtain an ob / ob obese mouse model of atherosclerosis.

2. The method for constructing an ob / ob obese mouse model of atherosclerosis according to claim 1, characterized in that: The body weight of the ob / ob mice is 40-50 g / mouse.

3. The method for constructing an ob / ob obese mouse model of atherosclerosis according to claim 1, characterized in that: The ob / ob mice were 8-week-old male ob / ob mice.

4. The method for constructing an ob / ob obese mouse model of atherosclerosis according to claim 3, characterized in that: The method for breeding 8-week-old male ob / ob mice comprises: raising the male ob / ob mice under pathogen-free conditions of 19-23°C, 40%-60% relative humidity, and repeatedly carrying out a cycle of 12 hours of light breeding and 12 hours of dark breeding until the mice are 8 weeks old, thereby obtaining 8-week-old male ob / ob mice.

5. The method for constructing an ob / ob obese mouse model of atherosclerosis according to claim 1, characterized in that: The injection volume of the AAV8-PCSK9 virus is 1×10^11 vg / mouse.

6. The method for constructing an ob / ob obese mouse model of atherosclerosis according to claim 1, characterized in that: The high-fat feed is selected from the feed with product number D12109C from Research Diets.

7. An ob / ob obese mouse model of atherosclerosis, characterized in that: The ob / ob obese mouse model of atherosclerosis is constructed by the method for constructing the ob / ob obese mouse model of atherosclerosis according to any one of claims 1 to 8.

8. Use of the method for constructing an ob / ob obese mouse model of atherosclerosis according to any one of claims 1 to 6 in studying the relationship between obesity and atherosclerosis.

9. The use according to claim 8, characterized in that: The ob / ob obese mouse model of atherosclerosis constructed by the construction method is used to develop the regulatory mechanism of obesity on atherosclerosis.

10. The use according to claim 8, characterized in that: The ob / ob obese mouse model of atherosclerosis constructed by the construction method is used to develop therapeutic targets and therapeutic drugs for obesity-related atherosclerosis.

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