Construction method of gene mouse for rapid modeling of advanced atherosclerotic vulnerable plaque

Abstract

The invention discloses a construction method of an atherosclerotic advanced vulnerable plaque rapid modeling gene mouse, and belongs to the technical field of biology. According to the method, an apolipoprotein E gene (apolipoprotein E, ApoE) knockout mouse which does not need to be subjected to induction and is specifically knocked out from an insulin-like growth factor 1 (IGF1) gene tissue is obtained through a hybridization method and a Cre-LoxP technology. According to the invention, a mononuclear phagocyte system specific knockout IGF1 gene is introduced into an ApoE- / -mouse genome for the first time, the characteristics of two genotypes are combined, and atherosclerotic advanced vulnerable plaque with the characteristics of large lipid core, a large number of inflammatory cells, typical thin fibrous caps, negative vascular remodeling and the like can be formed without induction; and the method is simple and easy to implement and good in repeatability, and the molding time is shortened.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for constructing a gene-based mouse model of vulnerable plaques in the late stage of atherosclerosis. Background Technology

[0002] Currently, cardiovascular disease is the leading cause of death from non-communicable diseases, and its disease burden and harm are increasing, making it a major public health issue. The primary cause of cardiovascular death is coronary heart disease, with atherosclerosis (AS) as the main pathological process. Research on atherosclerosis has become a hot topic in the cardiovascular field in recent years. Vulnerable plaque rupture is the leading cause of adverse cardiovascular events; it has been reported that more than two-thirds of acute myocardial infarctions originate from plaque rupture. However, due to differences between animal and human genomes, as well as the different pathophysiological processes of plaque formation, the current animal models of vulnerable plaques have long creation cycles and differ significantly from human vulnerable plaques. Therefore, establishing rapid animal models similar to human atherosclerotic plaques is a pressing problem that needs to be solved.

[0003] Currently, the main mouse model of atherosclerosis used in scientific research is the apolipoprotein E gene knockout mouse. ApoE - / - ) and low-density lipoprotein receptor gene knockout mice (ldlr - / - All of these methods require 8-week-old adult mice to be fed a high-fat diet for 16 weeks to establish an animal model of late-stage vulnerable atherosclerotic plaques. The experimental cycle is long and the research cost is high. Currently, there is a lack of a rapid and simple animal modeling method that conforms to the characteristics of vulnerable plaques in human research on vulnerable atherosclerotic plaques.

[0004] Currently, the main methods for constructing models for atherosclerosis research are as follows: 1. High-fat diet induction: Due to the differences between animal and human genomes, even under high-fat diet conditions, animals such as mice and rabbits are still unlikely to develop atherosclerosis. Only animals with highly homologous genomes to humans, such as monkeys and pigs, can produce plaques similar to those in humans. However, these animals are expensive, require long feeding times, and are difficult to manipulate, becoming a major limitation of research. 2. Vascular mechanical injury: Mechanically damaging the vascular endothelium can shorten plaque formation time, but the model is unstable, has poor reproducibility, and cannot simulate the entire pathophysiological process of plaque formation in humans. 3. Gene knockout technology: Gene knockout technology has been widely used in atherosclerosis research. Mice are the most widely used animals for gene knockout technology due to their rapid reproduction rate, short feeding time, and ease of manipulation. Currently, gene knockout mice widely used in atherosclerosis research include: ApoE - / - mice and LDLR - / -Mice, normal diet, ApoE - / - Mice can also form atherosclerotic plaques, but their cycle is long, atherosclerotic plaque formation is slow, and high-fat diet-fed ApoE - / - And LDLR - / - The formation cycle of atherosclerotic plaques in mice is shortened to within six months, and there is still a long cycle disadvantage, and it is difficult to observe the outward remodeling of human vulnerable plaques. Based on the above limitations, some researchers have used gene knockout technology to construct apolipoprotein E gene knockout dogs (publication number CN106987604A), which has the advantages of retaining the original symptoms of the disease and having a long disease phenotype duration, but due to the long feeding time of dogs, slow reproduction and other problems, it is not conducive to general research. The method of establishing a vulnerable plaque model by freezing blood vessels with liquid nitrogen (publication number CN101480359A) is not conducive to the study of the mechanism of vulnerable plaque formation and the exploration of intervention methods. The applicant obtained an atherosclerotic vulnerable plaque model animal (publication number CN117165631B) with inducible specific smooth muscle tissue knockout of the insulin-like growth factor 1 (IGF1) gene by hybridization and Cre-Loxp recombination technology, which carries the Y chromosome and needs to be induced to form a model, which has certain limitations and is not conducive to its popularization and application. IGF1 SUMMARY

[0005] The purpose of the present application is achieved by the following technical measures: A method for constructing a gene mouse of atherosclerotic late-stage vulnerable plaque rapid modeling, which is obtained by hybridization and Cre-LoxP recombination technology and has no need for induction IGF1 Gene tissue-specific knockout ApoE - Gene knockout Cre-LoxP mouse, the no-induction IGF1 Gene tissue-specific knockout means that the mouse can specifically knockout the mononuclear phagocyte system IGF1 gene without induction.

[0006] Preferably, the tissue-specific IGF1 Gene conditional knockout means that [Cx3cr1-iCre] [IGF1 flox ] .

[0007] Preferably, the construction method comprises the following steps: (1) Cross C57BL / 6 mice carrying IGF1-loxP recombinant genes with homologous ApoE gene knockout mice to obtain mice with genotype IGF1 flox / - ApoE ​+ / - F1 generation mice were self-crossed to obtain F2 generation mice. The F2 generation genotype was: IGF1 flox / flox ApoE - / - 、 IGF1 flox / flox ApoE + / - , IGF1 flox / flox , IGF1 flox / - ApoE + / - , IGF1 flox / - ApoE - / - , IGF1 flox / - , ApoE + / - 、 ApoE - / - or C57BL / 6; (2) Homozygous mice expressing Cx3cr1-iCre specifically from the homologous mononuclear phagocyte system were crossed with ApoE knockout mice to obtain F1' generation mice. The genotype of the F1' generation mice was: Cx3cr1-iCre + / - ApoE + / - F2' generation mice were obtained through self-pollination. The genotype of the F2' generation mice was: Cx3cr1-iCre + / + ApoE - / - , Cx3cr1-iCre + / + ApoE + / - , Cx3cr1- + / + iCre , Cx3cr1-iCre + / - ApoE - / - , Cx3cr1-iCre + / - ApoE + / - , Cx3cr1-iCre + / - , ApoE + / - , ApoE - / - Or C57BL / 6; (3) Select the genotype from step (1) that is IGF1 flox / flox ApoE - / - F2 generation mice and step (2) Cx3cr1- iCre+ / + ApoE - / - F2' generation mice are crossed with F2' generation mice of genotype Cx3cr1-iCre + / - IGF1 flox / - ApoE - / - F4 generation mice are obtained by self-crossing F3 generation mice, and the genotype of the F4 generation mice is Cx3cr1-iCre + / + IGF1 flox / flox ApoE - / - 、 Cx3cr1-iCre + / + IGF1 flox / - ApoE - / - , Cx3cr1-iCre + / + ApoE - / - , Cx3cr1-iCre + / - IGF1 flox / flox ApoE - / - , Cx3cr1-iCre + / - IGF1 flox / - ApoE - / - , Cx3cr1-iCre + / - ApoE - / - , IGF1 flox / flox ApoE - / - 、 IGF1 flox / - ApoE - / - or ApoE - / - ; (4) Mice with genotype Cx3cr1-iCre + / + IGF1 flox / flox ApoE - / - or Cx3cr1-iCre + / - IGF1 flox / flox ApoE - / - are fed with high-fat feed for 8 weeks to obtain atherosclerosis late-stage vulnerable plaque rapid modeling mouse.

[0008] Preferably, the high cholesterol and high fat feed is formulated as follows: 25% w / w of lard, 26% w / w of casein, 2% w / w of cholesterol and 0.22% w / w of choline hydroxide are added to the basic mouse feed.

[0009] Further, the present application also provides a method for constructing the gene mouse of the late vulnerable plaque of atherosclerosis, and the application of the gene mouse in preparing a model for researching the late vulnerable plaque of atherosclerosis.

[0010] [Cx3cr1-iCre][IGF1 flox ] is IGF1 a monocyte phagocyte system specific knockout of the gene, IGF1 is an endocrine and paracrine growth factor, which plays an important role in the progression of atherosclerosis (AS) by affecting the phenotype and function of various components of AS plaque, including macrophages, smooth muscle cells (SMC), endothelial cells and collagen. Poor expression of IGF-1 may lead to apoptosis of macrophages, SMC and endothelial cells, collagen decomposition, thinning of the fibrous cap, expansion of the lipid core, and increased vulnerability of the AS plaque, thus leading to plaque vulnerability, plaque rupture and acute coronary events. The applicant's previous research has found that the decrease of IGF1 level is closely related to the rupture of the culprit plaque and the increase of the necrotic lipid core in ST segment elevation myocardial infarction, further confirming that IGF1 gene plays an important role in promoting the stability of atherosclerotic plaques.

[0011] Therefore, the knockout of IGF1 gene can affect the structure of the blood vessel wall by affecting the components of the AS plaque, thus promoting the formation of atherosclerotic plaques. In addition, IGF1 double knockout mice (genotype IGF1 - / - are often unable to be born or die soon after birth due to severe genetic defects, therefore, the present application is based on ApoE - / - specific monocyte phagocyte system tissue knockout mice constructed by combining the mouse background with the Cre-LoxP recombination technology IGF1 double knockout mice (genotype [Cx3cr1-iCre][IGF1 flox ] ), the mice can develop and survive without induction, and only the monocyte phagocyte system knockout IGF1 gene is knocked out, thereby regulating the construction of the atherosclerotic plaque model. ApoEis a component of very low density lipoprotein (VLDL) and high density lipoprotein (HDL) and is involved in the transport of cholesterol. In contrast to the human lipoprotein profile, HDL is higher and low density lipoprotein (LDL) is lower in mice, and the cholesterol is mainly present in HDL.

[0012] knockout ApoE After knocking out the gene, cholesterol is mainly distributed in VLDL, and the large amount of cholesterol carried by VLDL cannot be combined with the cell surface lipoprotein receptor for degradation, resulting in accumulation and causing atherosclerosis. Previous studies have confirmed that, ApoE knockout mice are a spontaneous atherosclerosis model that is most similar to the progression of human atherosclerosis, and can accelerate the progression of atherosclerotic plaques after high-fat feeding. However, even if the progression of atherosclerosis is accelerated, the formation of vulnerable plaques requires more than 16 weeks of high-fat feeding, so the present application first uses Cre-loxP recombination technology to [Cx3cr1-iCre] [IGF1 flox ] recombination gene introduction ApoE - / - The mouse genome combines the characteristics of two gene knockout mice, and successfully constructs an atherosclerotic plaque that forms a large necrotic core, a large number of inflammatory cells, a thin fibrous cap, negative remodeling and other characteristics of human vulnerable plaques without induction after 8 weeks of high-fat feeding.

[0013] Due to the adoption of the above technical solutions, compared with the prior art, the present application has the following advantages: 1. The mouse model established according to the method of the present application can form a late-stage vulnerable plaque with a large lipid core, a large number of inflammatory cells, a typical thin fibrous cap, and vascular negative remodeling, shortens the modeling cycle, and better simulates the pathophysiological process of human vulnerable plaque formation, reduces the gap between animal experiments and the actual human body, provides a good animal basis for drug research and improvement of instrument consumables, and provides a good platform for exploring the mechanism of atherosclerosis. Using this platform, researchers can better explore the characteristics of vulnerable plaques and their intervention methods.

[0014] 2. The method of the present application is simple and easy to operate, and the modeling cycle is short. The mouse model established according to the method of the present application is a gene knockout mouse with C57BL / 6 mouse as the background, which has the advantages of high repeatability and easy breeding, and provides a good animal basis for the research of atherosclerotic vulnerable plaques.

[0015] The present application will be further described below in conjunction with the drawings and specific embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 A schematic diagram of the construction of a rapid atherosclerosis modeling mouse.

[0017] Figure 2 IGF1-CKO gene recombination process mode chart for Cre-Loxp recombination technology.

[0018] Figure 3 Genotype construction Cx3cr1-iCre + / + IGF1 flox / flox ApoE - / - And Cx3cr1-iCre + / - IGF1 flox / flox ApoE - / - Mouse verification result chart; Figure 3 A is the identification result of different gene recombination mice in the middle; Figure 3 B is Cx3cr1-iCre + / + IGF1 flox / flox ApoE - / - And Cx3cr1-iCre + / - IGF1 flox / flox ApoE - / - Mouse gene electrophoresis verification result chart.

[0019] Figure 4 Aortic gross oil red O staining chart of model mice and control group mice after high-fat feeding; Figure 4 A is the genotype of the mice after 8 weeks of high-fat feeding Cx3cr1-iCre + / + IGF1 flox / flox ApoE - / - Aorta of mice, Figure 4 B is the genotype of the mice after 8 weeks of high-fat feeding Cx3cr1-iCre + / - IGF1 flox / flox ApoE - / - Aorta of mice, Figure 4 C and Figure 4 D is the genotype of the mice after 16 weeks of high-fat feeding ApoE - / - Aorta of mice.

[0020] Figure 5 HE staining chart of aortic root frozen section of model mice and control mice; Figure 5 A is the genotype of the mice after 8 weeks of high-fat feeding Cx3cr1-iCre + / + IGF1 flox / flox ApoE- / - HE staining of frozen sections of the aortic root of mice after 8 weeks of high-fat feeding; negative vascular remodeling and reduced lumen area. Figure 5 B is ApoE - / - HE staining of frozen sections of the aortic root of mice after 16 weeks of high-fat diet. C: necrotic nucleus, P: plaque, L: vascular lumen. The arrow points to the thinnest fibrous cap. Figure 5 C is Figure 5 A magnified view of the area within box A in the middle. Figure 5 D is Figure 5 A magnified view of the area within box B in the middle. Figure 5 A large number of inflammatory cells can be seen in the middle C. Figure 5 Inflammatory cells in the middle D are more Figure 5 There are few C-shaped diagrams.

[0021] Figure 6 Oil Red O staining images of the aortic root of model mice and control mice after high-fat feeding; Figure 6 A in the middle is Cx3cr1-iCre + / + IGF1 flox / flox ApoE - / - Oil Red O staining of frozen sections of the aortic root of mice after 8 weeks of high-fat diet. Figure 6 B was fed a high-fat diet for 16 weeks. ApoE - / - Oil Red O staining image of a frozen section of the aortic root of a mouse.

[0022] Figure 7 This is a comparison table of measurement parameters between the control group and the model group. Detailed Implementation

[0023] Example: As attached Figure 1 To be continued Figure 6 As shown, mice with the IGF1 gene inserted into the LoxP site specifically recognized by Cre recombinase (genotype: IGF1 flox / flox (Ordered from Suzhou Cyagen Biotechnology Co., Ltd.) ApoE Gene knockout mice (genotype: ApoE - / - F1 mice were obtained by hybridization (purchased from Suzhou Cyagen Biotechnology Co., Ltd.), with the genotype being... IGF1 flox / - ApoE + / - DNA was extracted from tail tissue of F1 generation mice for gene identification. (The mouse tail genotype identification kit was: Rapid Mouse Tail Genotype Identification Kit: Beyotime D7283M).

[0024] Identify IGF1-LoxP genotype: tail DNA of mice was amplified by PCR, PCR system: 2x Taq PCR Master Mix: 12.5 μL, primer (use F1 / R1 to verify pure hybrid, confirm the genotype of mice is flox / + or flox / flox 10 μM (1 μL) each, genomic DNA 100 ng, add ddH2O to 25 μL, reaction conditions: 94℃ 3 min, 94℃ 30 sec, 55℃ 30 sec, 72℃ 1 min, 72℃ 5 min, cycle number is 35, identification primer (5'-3') is: CKO-F1 (5'-3'): -TGATTGGAAAGATCCAGTGTCCC CKO-R1 (5'-3'): -GCCCAGTCCTATCACCTTTCTTTT The amplified product was electrophoresed on agarose gel, and the identification results are shown in Figure 2 , pure hybrid (flox / flox) has only a single 339bp band, hybrid (flox / -) has 339bp and 271bp two bands, wild type has only a single 271bp.

[0025] 1. Cx3cr1-iCre specific expression of mononuclear phagocyte system and apolipoprotein E gene knockout homozygous mice were crossed, and F1' generation mice were obtained, the genotype of F1' generation mice was ApoE Cx3cr1-iCre + / - ApoE + / - ; F2' generation mice were obtained by self-crossing F1' generation mice, and the genotype was Cx3cr1-iCre + / + ApoE - / - , Cx3cr1-iCre + / + ApoE + / - , Cx3cr1- iCre + / + , Cx3cr1-iCre + / - ApoE - / - , Cx3cr1-iCre + / - ApoE + / - , Cx3cr1-iCre + / - , ApoE + / - , ApoE - / - or C57BL / 6; (tail genotype identification kit as above).

[0026] ​2. Identification of Cx3cr1-iCre genotype: tail DNA of mice was amplified by PCR, the PCR system was as follows: 2x Taq PCR Master Mix: 12.5 μL, primers (F1 / R1 was used for verification, to confirm whether the genotype of mice was homozygous Cx3cr1-iCre + / + ; F2 / R1 was used for verification, to confirm whether the genotype of mice was wild Cx3cr1-iCre - / - ) 10 μM (1 μL) each, genomic DNA 100 ng, add ddH2O to 25 μL, reaction conditions: 94°C for 3 min, 94°C for 30 sec, 55°C for 30 sec, 72°C for 1 min, 72°C for 5 min, cycle number was 35, the identification primer (5'-3') was as follows: CX3CR1-Cre-F1 (5'-3'): -GACATTTGCCTTGCTGGAC CX3CR1-Cre-F2 (5'-3'): -CCTCAGTGTGACGGAGACAG CX3CR1-Cre-R1 (5'-3'): -GCAGGGAAATCTGATGCAAG The amplification product was electrophoresed on agarose gel, and the identification results were as follows: Figure 3 The homozygote (cre / cre) had only a single 380 bp band, the heterozygote (cre / -) had two 380 bp and 302 bp bands, and the wild type (- / -) had only a single 302 bp band.

[0027] 3. F2 generation mice with genotype IGF1 flox / flox ApoE - / - were crossed with F2' generation mice with genotype Cx3cr1-iCre + / + ApoE - / - to obtain F3 generation mice, and the genotype of F3 generation mice was Cx3cr1-iCre + / - IGF1 flox / - ApoE - / - F4 generation mice were obtained by self-crossing F3 generation mice, and the genotype of F4 generation mice was Cx3cr1-iCre + / + IGF1 flox / flox ApoE - / - , Cx3cr1-iCre + / + IGF1 flox / - ApoE - / - , Cx3cr1-iCre+ / + ApoE - / - , Cx3cr1-iCre + / - IGF1 flox / flox ApoE - / - , Cx3cr1-iCre + / - IGF1 flox / - ApoE - / - , Cx3cr1-iCre + / - ApoE - / - , IGF1 flox / flox ApoE - / - , IGF1 flox / - ApoE - / - or ApoE - / - DNA was extracted from the tails of F4 generation mice, and the genotypes of the mice were identified Cx3cr1-iCre, IGF1-flox and ApoE genotypes (mouse tail genotype identification kit as above).

[0028] 4. ApoE genotype identification: PCR amplification was performed on the mouse tail DNA, and the PCR system was as follows: 2x Taq PCR Master Mix: 12.5 μL, primer (ApoE-c common primer, ApoE-m mutant primer, ApoE-w wild type primer) 10 μM (1 μL) each, genomic DNA 100 ng, and ddH2O to 25 μL. The reaction conditions were as follows: 94°C for 3 min, 94°C for 30 sec, 55°C for 30 sec, 72°C for 1 min, 72°C for 5 min, and the cycle number was 35. The identification primer (5'-3') was as follows: ApoE-c (5'-3'): -GCCTAGCCGAGGGAGAGCCG ApoE-w (5'-3'): -TGTGACTTGGGAGCTCTGCAGC ApoE-m (5'-3'): -GCCGCCCCGACTGCATCT The amplification product was subjected to agarose gel electrophoresis, and the identification results were as follows: Figure 3 , ApoE - / - The homozygous mouse can amplify a single band of 245 bp in length, the normal wild type mouse can amplify a single band of 155 bp in length, and the heterozygous type can amplify bands of 245 bp and 155 bp at the same time.

[0029] 5. The genotype of the F3 generation mouse selected above is Cx3cr1-iCre + / +IGF1 flox / flox ApoE - / - or Cx3cr1-iCre + / - IGF1 flox / flox ApoE - / - ApoE- / - mice are ideal vulnerable plaque model. The genotype of ApoE- / - mice is ApoE- / -. Cx3cr1-iCre + / - IGF1 flox / - ApoE - / - and Cx3cr1-iCre + / - IGF1 flox / - ApoE - / - of 8 The model group mice of 8 weeks old were fed with high-fat diet for 8 weeks and the control group mice of the same age were fed with normal diet. ApoE - / - After the mice were fed with high-fat diet (western-type-diet, WD) for 8 weeks and 16 weeks, the hearts of the mice were quickly frozen in liquid nitrogen, and the frozen sections were embedded in OCT and fixed in cold acetone. HE staining, Masson staining and oil red O staining were performed, respectively, and the data were analyzed by ImageJ. The areas of plaques and components were counted, and the results are shown in Figures 4-7 The high-fat diet formula was prepared by adding 25% w / w of lard, 26% w / w of casein, 2% w / w of cholesterol and 0.22% w / w of choline hydroxide tartrate into the basic diet for mice. The improved XT108C (2% cholesterol) was purchased from Jiangsu Cooperation Pharmaceutical and Biological Engineering Co., Ltd.

[0030] 6. Aortic gross oil red O staining: After the sample was taken, it was fixed in 4% paraformaldehyde for 24 hours. The blood vessel was cut open, washed with PBS for 3 times, 5 min each time, stained with oil red O for 15 min, and washed with 75% alcohol until the normal tissue turned white. The sample was fixed and photographed. The results of gross staining showed that there was no significant difference in the degree of atherosclerosis between the model group mice fed with high-fat diet for 8 weeks and the control group mice fed with high-fat diet for 16 weeks, as shown in Figure 4 .

[0031] The model group mice fed with high-fat diet for 8 weeks Cx3cr1-iCre + / + IGF1 flox / flox ApoE - / - or Cx3cr1-iCre + / - IGF1 flox / flox ApoE - / -The mice gradually appeared extravascular remodeling, the lumen area increased, the necrotic core became larger, the fibrous cap became thinner, and a large number of inflammatory cell infiltrations were visible, and Figure 5 Compared with the mice fed with high-fat diet for 16 weeks ApoE - / - The necrotic core was larger, and there was no significant difference.

[0032] Conclusion: The mouse model established by the method can quickly form a vulnerable plaque with characteristics of a large lipid core, a large number of inflammatory cells, a typical thin fibrous cap, extravascular remodeling and the like, which provides a good platform for exploring the mechanism of atherosclerosis. Using this platform, researchers can better explore the characteristics of vulnerable plaques and their intervention methods.

Claims

1. A method for constructing a gene mouse of rapid modeling of late-stage vulnerable atherosclerotic plaque, characterized in that, Insulin-like growth factor 1 (IGF-1) was obtained without induction using hybridization and Cre-loxp recombination technology. IGF1 Apolipoprotein E gene knockout Cre-loxp mice were obtained through tissue-specific knockout of the gene, specifically by hybridization and Cre-loxp recombination technology, possessing insulin-like growth factor 1 (IGF-1) without induction. IGF1 The tissue-specific knockout of the apolipoprotein E gene in Cre-loxp mice refers to mice that can specifically knock out the apolipoprotein E gene in the mononuclear phagocyte system without induction. IGF1 Gene; The construction method comprises the following steps: Step (1) C57BL / 6 mice carrying IGF1-loxP recombinant gene are crossed with homozygous ApoE gene knockout mice to obtain F1 generation mice with genotype IGF1 flox / - ApoE + / - F1 of ApoE F2 generation mice, C57BL / 6 mice carrying IGF1-loxP recombinant gene are inbred to obtain F2 generation mice; Step (2) cross the Cx3cr1-iCre homozygous mice with specific expression of mononuclear phagocyte system homologous with ApoE Cross the gene knockout mice to obtain F1' generation mice, cross the Cx3cr1-iCre homozygous mice with specific expression of mononuclear phagocyte system homologous with Step (3) cross the F2 generation mice in step (1) with the F2' generation mice in step (2) to obtain F3 generation; self-cross the F3 generation mice to obtain F4 generation mice; Step (4) select the F4 generation mice in step (3), and feed the mice with high-fat feed for 8 weeks to obtain the atherosclerosis late vulnerable plaque rapid modeling mice.

2. The method for constructing a gene mouse of rapid modeling of late vulnerable atherosclerotic plaque according to claim 1, wherein: The F2 generation of mice is genotyped for IGF1 flox / flox ApoE - / - IGF1 flox / flox ApoE + / - IGF1 flox / flox 、 IGF1 flox / - ApoE + / - IGF1 flox / - ApoE - / - IGF1 flox / - ApoE + / - ApoE - / - or C57BL / 6.

3. The method for constructing a gene mouse of rapid modeling of late vulnerable atherosclerotic plaque according to claim 2, wherein: The F1' generation mice have a genotype of Cx3cr1-iCre + / - ApoE + / -- The F2' generation mice have a genotype of Cx3cr1-iCre + / + ApoE - / - Cx3cr1-iCre + / + ApoE + / - Cx3cr1-iCre + / + Cx3cr1-iCre + / - ApoE - / - 、 Cx3cr1-iCre + / - ApoE + / - Cx3cr1-iCre + / - ApoE + / - ApoE - / - or C57BL / 6.

4. The method for constructing a gene mouse of rapid modeling of late vulnerable atherosclerotic plaque according to claim 3, wherein: The genotype of the F2 generation female mice in the step (3) is IGF1 flox / flox ApoE - / - The genotype of the F2' generation male mice in the step (3) is Cx3cr1-iCre + / + ApoE - / - The genotype of the F3 generation mice is Cx3cr1-iCre + / - IGF1 flox / - ApoE - / - .

5. The method for constructing a mouse model of rapid progression of late vulnerable atherosclerotic plaque according to claim 4, wherein: The genotype of the F4 generation rats in the step (3) is Cx3cr1-iCre + / + IGF1 flox / flox ApoE - / - 、 Cx3cr1-iCre + / + IGF1 flox / - ApoE - / - Cx3cr1-iCre + / + IGF1 - / - ApoE + / - Cx3cr1-iCre flox / flox IGF1 - / - ApoE + / - Cx3cr1-iCre flox / - IGF1 - / - ApoE + / - Cx3cr1-iCre - / - IGF1 flox / flox ApoE - / - 、 Cx3cr1-iCre flox / - IGF1 - / - ApoE - / - .

6. The method for constructing a gene mouse of rapid modeling of late vulnerable atherosclerotic plaque according to claim 5, wherein: The genotype of the F4 generation rats in the step (4) is Cx3cr1-iCre + / + IGF1 flox / flox ApoE - / - or Cx3cr1-iCre + / - IGF1 flox / flox ApoE - / - .

7. The method for constructing a mouse model of rapid progression of late vulnerable atherosclerotic plaque according to claim 1, wherein: Cx3cr1-iCre IGF1 ApoE The formula of the high-fat feed is that: 25% w / w of lard, 26% w / w of casein, 2% w / w of cholesterol and 0.22% w / w of choline hydroxide are added into the mouse basic feed.

8. The use of the construction method of the atherosclerosis late vulnerable plaque rapid modeling gene mouse in the preparation of a model for researching atherosclerosis late stage and vulnerable plaque.

Images