Construction method and application of mouse model with fat dystrophy complicated with atherosclerosis

Abstract

The invention relates to the technical field of animal models, and particularly discloses a construction method and application of a mouse model with fat dystrophy complicated with atherosclerosis, and the construction method comprises the following steps: mating MED1flox / flox mice with Adipoq-CreERT2 mice to obtain F1-generation mice; the F1-generation mice and MED1flox / flox mice are subjected to backcross, the mice with the genotype being MED1fl / flAdipoq-Cre < + / -> are screened, tamoxifen is injected to induce fat cell specific Cre recombinase expression, and fat cell specific MED1 gene knockout mice are obtained; and performing caudal vein injection of AAV8-PCSK9 virus and feeding with a high-cholesterol and high-fat feed to obtain the fat dystrophy complicated with atherosclerosis mouse model. According to the method provided by the invention, the mouse model with fat dystrophy complicated with atherosclerosis is successfully constructed and obtained, and collaborative simulation and regulation of phenotypes of two diseases are realized.

Description

Technical Field

[0001] This invention relates to the field of animal model technology, specifically to a method for constructing and applying a mouse model of lipodystrophy complicated with atherosclerosis. Background Technology

[0002] Lipodystrophy syndromes (LDS) are a group of rare metabolic diseases characterized by abnormal distribution, absence, or dysfunction of adipose tissue. In LDS patients, the loss of lipid storage function leads to lipid "overflow" into non-adipose tissues (such as the liver and muscles), causing severe insulin resistance, hypertriglyceridemia, fatty liver, and early-onset atherosclerosis (AS). The latter is a leading cause of death in these patients. To further explore its pathological mechanisms and develop effective treatment strategies, it is crucial to construct animal models that accurately simulate lipodystrophy complicated by atherosclerosis.

[0003] Currently, the main models of lipodystrophy include gene-edited mouse models, such as Agpat2. - / - mice 、 Bscl2 - / - mice 、 Plin1 - / - Models such as mice and adipocyte PPARγ-specific knockout mice exhibit severe metabolic disturbances, but the degree of spontaneous atherosclerosis is mild or atypical. The main atherosclerosis models include ApoE. - / - Mice and LDLR - / - Mice and other mice fed a high-fat diet can develop typical atherosclerotic plaques, but these mouse models retain intact fat storage capacity, and their metabolic background is distinctly different from that of human lipodystrophy. Existing methods for constructing animal models only generate single-disease models. Since human diseases are often complex processes involving the interaction of multiple pathologies, a single-disease model cannot adequately simulate the rapid and aggressive atherosclerotic pathological environment driven by "lipid spillover" faced by patients with lipodystrophy. Therefore, there is an urgent need in this field to provide a method for constructing animal models that highly simulate lipodystrophy complicated by atherosclerosis. Summary of the Invention

[0004] To develop a method for constructing a mouse model of lipodystrophy complicated with atherosclerosis, this invention provides a method for constructing and applying such a mouse model. The method provided by this invention successfully constructs a mouse model of lipodystrophy complicated with atherosclerosis, achieving synergistic simulation and regulation of the two disease phenotypes, and providing a basic model for screening drugs for the treatment or prevention of lipodystrophy complicated with atherosclerosis.

[0005] This invention provides a method for constructing a mouse model of lipodystrophy complicated with atherosclerosis, comprising the following steps: MED1 flox / flox Mice and Adipoq-CreER T2 Mice were mated to obtain the MED1 genotype. fl / + / Adipoq-Cre + / - F1 generation mice; F1 generation mice were compared with MED1 flox / flox Backcrossing mice yielded F2 generation mice, with the MED1 genotype being [missing information]. fl / fl / Adipoq-Cre + / - MED1 gene knockout was achieved in mice induced by tamoxifen, resulting in adipocyte-specific MED1 gene knockout (MED1). ΔAdi Mouse model; MED1 ΔAdi A mouse model of lipodystrophy and atherosclerosis was obtained by injecting AAV8-PCSK9 virus into the tail vein and feeding the mouse with a high-cholesterol diet.

[0006] The method provided by this invention successfully constructed a mouse model of lipodystrophy complicated with atherosclerosis, realizing the synergistic simulation and regulation of the two disease phenotypes, and providing a basic model for screening drugs for the treatment or prevention of lipodystrophy complicated with atherosclerosis.

[0007] Furthermore, before tamoxifen induction, the MED1 conditional knockout model mice were obtained from F2 generation mice with the MED1 genotype. fl / fl / Adipoq-Cre + / - Mice and genotype MED1 fl / fl / Adipoq-Cre - / - Mice were paired and bred to establish a stable genetic breeding line. Half of the offspring mice from this stable genetic breeding line were MED1 conditional knockout model mice, which were used as tamoxifen-induced experimental mice.

[0008] Furthermore, the tamoxifen induction is performed via intraperitoneal injection at a dose of 25 mg / kg body weight to 50 mg / kg body weight.

[0009] Furthermore, the tail vein injection of AAV8-PCSK9 virus dose is 1×10⁻⁶. 12 VG~1×10 13 VG.

[0010] Furthermore, the diet was continued for 12 to 14 weeks with a high-fat, high-cholesterol diet.

[0011] The present invention also provides the application of a mouse model of lipodystrophy complicated with atherosclerosis in screening drugs for the prevention or treatment of lipodystrophy complicated with atherosclerosis, wherein the mouse model of lipodystrophy complicated with atherosclerosis is constructed by the above-described construction method.

[0012] Furthermore, the application involves administering candidate drugs during the construction of a mouse model of lipodystrophy complicated with atherosclerosis, and screening for candidate drugs that can simultaneously improve fat function, correct metabolic disorders, and inhibit or reverse atherosclerotic plaques.

[0013] Furthermore, the dosing strategy for screening drugs to prevent lipodystrophy complicated with atherosclerosis was as follows: administration was performed simultaneously with the initiation of tail vein injection of AAV8-PCSK9 virus and feeding of a high-cholesterol diet, and the drug's ability to prevent disease was evaluated.

[0014] Furthermore, the drug administration strategy for screening drugs to treat lipodystrophy complicated with atherosclerosis was as follows: administration was started 4 weeks after tail vein injection of AAV8-PCSK9 virus and feeding with a high-cholesterol diet, and continued for 8 to 12 weeks.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: The method provided by this invention successfully constructed a mouse model of lipodystrophy complicated with atherosclerosis, achieving synergistic simulation and regulation of the two disease phenotypes.

[0016] Specifically, firstly, this invention employs Cre-LoxP conditional gene knockout technology, combined with tamoxifen induction, to achieve specific and time-controlled knockout of the MED1 gene in adipose tissue. This not only avoids the non-specific metabolic abnormalities and systemic developmental defects caused by traditional systemic gene knockout or random transgenic methods, but also significantly improves the model's reproducibility and the reliability of experimental data. Secondly, this invention constructs a mouse model of lipodystrophy combined with atherosclerosis. Compared with single models, this model has multiple advantages in its technical approach. The inducible gene knockout strategy minimizes interference from non-target phenotypes, shortens model establishment time, and reduces animal husbandry costs. Simultaneously, the model exhibits strong phenotypic consistency and minimal individual differences, providing an ideal preclinical evaluation platform for efficiently and reliably screening therapeutic drugs (such as leptin replacement therapy and PPARγ agonists). Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 MED1 ΔAdi Schematic diagram of mouse model construction and validation; In the diagram, A represents MED1. ΔAdi A schematic diagram illustrating the construction of the mouse model; B is MED1 ΔAdi A schematic diagram of the deletion of exons 8-10 of the MED1 gene in a mouse model; C is MED1 ΔAdi Genotyping results of the mouse model; D is MED1 ΔAdi Changes in MED1 mRNA expression levels in various adipose tissues of a mouse model.

[0019] Figure 2 MED1 ΔAdi Mouse body composition analysis; In the diagram, A represents MED1. ΔAdi Mice and their littermates MED1 fl / fl Comparison of mouse morphology (control group); B is MED1 ΔAdi Mice and their littermates MED1 fl / fl Comparison of body weight of mice (control group); C is MED1 ΔAdi Mice and their littermates MED1 fl / fl Comparison of the percentage of fat and lean meat in body weight of mice (control group).

[0020] Figure 3 MED1 ΔAdi Weight and gross observation of mouse adipose tissue; In the image, A represents a male MED1. ΔAdi Changes in SAT, VAT, and BAT in mice; B is female MED1 ΔAdi Changes in SAT, VAT, and BAT in mice.

[0021] Figure 4 MED1 ΔAdi Mouse histological staining images and statistical analysis; In the diagram, A represents MED1. ΔAdiH&E staining results of mouse white adipose tissue (SAT) (left image), male MED1 ΔAdi Changes in white adipose tissue (SAT) in mice (middle image) and in female MED1 ΔAdi Changes in mouse white adipose tissue (SAT) (right figure); B is MED1 ΔAdi H&E staining results of mouse white adipose tissue (VAT) (left image), male MED1 ΔAdi Changes in white adipose tissue (VAT) in mice (middle image) and female MED1 ΔAdi Changes in mouse white adipose tissue (VAT) (right figure); C is MED1 ΔAdi H&E staining results of brown adipose tissue (BAT) in mice (left image), and male MED1 ΔAdi Changes in brown adipose tissue (BAT) in mice (middle image) and in female MED1 ΔAdi Changes in brown adipose tissue (BAT) in mice (right figure).

[0022] Figure 5 MED1 ΔAdi Changes in blood lipid levels in a mouse model of atherosclerosis; In the figure, A represents the changes in total cholesterol (TC) levels in an adipose tissue MED1-specific knockout mouse model of atherosclerosis. B is MED1 ΔAdi Changes in triglyceride (TG) levels in a mouse model of atherosclerosis.

[0023] Figure 6 MED1 ΔAdi Mapping and statistics of atherosclerotic plaques in a mouse model of atherosclerosis; In the figure, A represents the control group (left) and mouse MED1. ΔAdi A mouse model of atherosclerosis (right): Atherosclerotic plaques. B is MED1 ΔAdi Overall statistical diagram of atherosclerotic plaques in the aorta of a mouse model of atherosclerosis.

[0024] C is MED1 ΔAdi Statistical diagram of atherosclerotic plaque lesions in the aortic arch, thoracic aorta, and abdominal aorta of a mouse model of atherosclerosis.

[0025] Figure 7 H&E and Oil Red O staining of aortic root sections and their statistical analysis results; In the figure, A shows the H&E and Oil Red O staining results of a section of the aortic root; B is a statistical chart of Oil Red O staining analysis of aortic root sections.

[0026] Figure 8 MED1 ΔAdi Body weight and weight of various adipose tissues in a mouse model of atherosclerosis; In the figure, A represents the control group mice and MED1 mice. ΔAdi Comparison of body weight in a mouse model of atherosclerosis; B represents the control group mice and MED1 mice. ΔAdi Statistics on liver weight and adipose tissue weight in a mouse model of atherosclerosis; C is MED1 ΔAdi Changes in liver weight ratio, SAT, VAT, and BAT in a mouse model of atherosclerosis.

[0027] Figure 9 MED1 ΔAdi Staining of adipose tissue sections from mice with an atherosclerosis model; In the figure, A shows the H&E staining results of adipose tissue sections; B is MED1 ΔAdi Immunohistochemical staining of CD68 macrophages in adipose tissue of a mouse model of atherosclerosis; C is MED1 ΔAdi Statistical analysis results of CD68-positive area in adipose tissue of mice with atherosclerosis model. Detailed Implementation

[0028] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0029] Information on the experimental reagents and mouse types and their sources used in this invention: Tamoxifen: Sigma-Aldrich, USA (Product No.: T5648-1G).

[0030] MED1 flox / flox The mice were donated by the Experimental Animal Center of Xi'an Jiaotong University.

[0031] Adipoq-CreER T2 The transgenic mice were donated by the Experimental Animal Center of Xi'an Jiaotong University and are referred to as Adipoq-Cre transgenic mice.

[0032] MED1 from the Experimental Animal Center of Xi'an Jiaotong University flox / flox Mice and Adipoq-CreER T2 All transgenic mice were given away free of charge. The AAV8-PCSK9 virus was purchased from Shandong Weizhen Biotechnology Co., Ltd., product number: WZP58376.

[0033] Example 1: A method for constructing a mouse model of lipodystrophy complicated with atherosclerosis.

[0034] I. Adipocyte-specific MED1 gene knockout mouse model (MED1 ΔAdi Construction of ) homozygous MED1 flox / flox Mice were mated with Adipoq-Cre transgenic mice to obtain the F1 generation of mice, whose genotype was MED1. fl / + Adipoq-Cre + / - F1 generation mice were compared with MED1 flox / flox Mice were backcrossed to obtain the F2 generation mouse population, which included mice with genotype MED1. fl / fl Adipoq-Cre + / - Mice and genotype MED1 fl / fl Adipoq-Cre - / - Mice. The F2 generation mice had the MED1 genotype. fl / fl Adipoq-Cre + / - In mice induced by tamoxifen, the MED1 gene can be specifically knocked out in adipocytes.

[0035] To establish a stable breeding population, MED1 conditional knockout model mice were bred with MED1 mice. fl / fl Adipoq-Cre - / - Mice are paired, meaning a stable genetic breeding line is established that can stably produce two types of offspring: half are MED1. fl / fl Adipoq-Cre + / - The experimental mice (MED1 conditional knockout model mice, which require induction to knock out), half of them are MED1. fl / fl Adipoq-Cre - / - (MED1) fl / fl The control mice provided ideal experimental material for subsequent research.

[0036] In MED1 conditional knockout mouse models, tamoxifen (50 mg / kg effective dose) was administered intraperitoneally to induce adipocyte-specific Cre recombinase expression, thereby achieving conditional knockout of the MED1 gene and obtaining an adipocyte-specific MED1 gene knockout mouse model (MED1). ΔAdi And with MED1 from the same litter. fl / fl Adipoq-Cre - / - (MED1) fl / fl Mice were used as a control. The specific steps are as follows:

[0037] 1. A Tamoxifen-induced adipocyte-specific MED1 gene knockout mouse model was constructed using the Cre-LoxP recombinase system. (1) Genetic hybridization and backcrossing steps: such as Figure 1 As shown in A, homozygous MED1 flox / flox MED1 was obtained by crossing mice with Adipoq-Cre mice and breeding them. fl / + Adipoq-Cre + / - Mice (F1 generation), F1 generation mice were compared with MED1 flox / flox Backcrossing homozygous mice to obtain F2 generation mice, and then screening for mice with genotype MED1 in the F2 generation. fl / fl Adipoq-Cre + / - MED1 conditional knockout mice and MED1 fl / fl Adipoq-Cre - / - (MED1) fl / fl Mice.

[0038] (2) Breeding steps: Use MED1 fl / fl Adipoq-Cre + / - (MED1 conditional knockout model mice) Mice and MED1 fl / fl Adipoq-Cre - / - Mice can be paired and bred to establish a stable genetic breeding line, yielding a population of offspring mice for experimentation. Half of the offspring population should be MED1 conditional knockout model mice (requiring induction), and the other half should be MED1 mice. fl / fl Adipoq-Cre - / - Control mice.

[0039] (3) Induction knockout steps: Dissolve Tamoxifen powder in corn oil to prepare a solution with a concentration of 20 mg / ml. After it is fully dissolved, store it at 4℃ and keep it away from light throughout the process. Use it within one week.

[0040] When MED1 conditional knockout mice were 6 weeks old, they were injected intraperitoneally with a single dose of 50 mg / kg body weight every 24 hours for 5 consecutive days. After the administration was completed, a 7-day interval was allowed to allow Cre recombinase to be fully activated and the gene knockout to be completed. At this point, the MED1 conditional knockout mice were considered to have become adipocyte MED1-specific knockout mice and were designated as MED1 mice. ΔAdi Mice. Littermate control mice MED1 fl / fl Adipoq-Cre - / - (MED1) fl / fl MED1-specific knockout mice were not subjected to knockout; therefore, two groups of mice were obtained after induction: the experimental group (MED1-specific knockout mice in adipocytes) and the control group (MED1-specific knockout mice). fl / fl Mice).

[0041] 2. Gene knockout analysis and genotype identification Gene knockout region characteristics such as Figure 1 As shown in B. The resulting knockout mice had exons 8-10 of the MED1 gene deleted.

[0042] Genotyping: Genomic DNA was extracted from the tail of model mice and used as a PCR template. PCR amplification was performed using the MED1-LoxP primer pair and the Adipoq-CreERT2 primer pair, yielding fragments of 1800 bp and 600 bp, respectively. Figure 1 As shown in Figure C, the amplification of a 1800 bp fragment indicates the presence of the flux allele, and the amplification of a 600 bp fragment indicates the presence of the Adipoq-CreERT2 recombinase gene. The simultaneous fulfillment of both conditions indicates that the MED1 gene has been knocked out and simultaneously carries the Cre recombinase gene, i.e., MED1. fl / fl Adipoq-Cre + / - (MED1 conditional knockout model mouse).

[0043] The MED1-LoxP primer pair includes the upstream primer shown in SEQ ID NO.1 and the downstream primer shown in SEQ ID NO.2. The Adipoq-CreERT2 primer pair includes the upstream primer shown in SEQ ID NO.3 and the downstream primer shown in SEQ ID NO.4.

[0044] SEQ ID NO.1: 5'-TCCATCTGACCTGCTGGATGATAA-3'; SEQ ID NO. 2: 5'-GGGTTGACCCCATAATT-3'.

[0045] SEQ ID NO.3: 5'-TGGTGCATCTGAAGACACTACA-3'; SEQ ID NO.4: 5'-TGCTGTTGGATGGTCTTCACAG-3'.

[0046] 3. RT-qPCR was used to verify the expression level of MED1 mRNA in adipose tissue of MED1-specific knockout mice obtained after Tamoxifen induction. RNA was extracted from primary adipocytes of three different sources—subcutaneous adipose tissue (SAT), visceral adipose tissue (VAT), and brown adipose tissue (BAT)—from MED1-specific knockout mice for RT-qPCR validation to confirm the MED1 knockout efficiency. The RT-qPCR primers are shown in Table 1, the reaction system in Table 2, and the reaction procedure in Table 3.

[0047] The results are as follows Figure 1 As shown in D, with MED1 fl / fl Compared with mice, the expression level of MED1 mRNA in adipocyte MED1-specific knockout mice was significantly downregulated compared with the control group, proving that MED1 in adipocytes was effectively knocked out and the gene knockout mice were successfully constructed.

[0048] 4. MED1-specific knockout mice in adipocytes (MED1) ΔAdi The model exhibited a lipodystrophy phenotype. (1) Changes in morphology and body weight of MED1-specific knockout mice in adipocytes Figure 2 Figure A shows 8-week-old male adipocyte MED1-specific knockout mice and female adipocyte MED1-specific knockout mice (experimental group) and their littermates. fl / fl Mice (control group) showed no significant difference in appearance. Figure 2 Furthermore, there was no statistically significant difference in body weight between the experimental and control groups (see A). Figure 2 (B). Body composition analysis results showed that, compared with the control group, MED1-specific knockout mice had significantly reduced fat mass and correspondingly increased lean mass (e.g., ...). Figure 2 (C).

[0049] (2) Changes in weight and distribution of adipose tissue in MED1-specific knockout mice Note: mean ± SEM.P <0.05,** P <0.01, *** P <0.001. 8-week-old male and female MED1 fl / fl and MED1 ΔAdi Differences in mouse adipose tissue (SAT, VAT, and BAT). In the Male group, MED1 fl / fl With MED1 ΔAdi The SAT showed a highly significant difference (***), the VAT showed a highly significant difference (**), and the BAT showed a significant difference (*). In the Female group, MED1... fl / fl With MED1 ΔAdi There was a highly significant difference in SAT (**), a significant difference in VAT (*), and no significant difference in BAT (ns).

[0050] After euthanasia of mice, white adipose tissue (SAT, VAT) and brown adipose tissue (BAT) were collected from both the experimental and control groups and weighed. Figure 3 As shown in A and Table 4, the tissue macroscopically reveals that in male MED1 ΔAdi In mice, SAT, VAT, and BAT were all significantly reduced, especially in female MED1 mice. ΔAdi In mice, white adipose tissue (SAT and VAT) was also significantly reduced, but BAT showed no significant difference (e.g., Figure 3 (As shown in B and Table 4). The above experimental data indicate that the absence of MED1 in adipose tissue can lead to atrophy of adipose tissue in mice, suggesting abnormal lipid metabolism.

[0051] (3) Pathological changes in adipose tissue of MED1-deficient mice Collect MED1 fl / fl and MED1 ΔAdi Adipose tissue from various parts of the mouse was embedded in paraffin, sectioned, and stained with H&E.

[0052] like Figure 4 A~ Figure 4 C10 shows the H&E staining results for SAT, VAT, and BAT, respectively. Compared to MED1... fl / fl Mice, MED1 ΔAdi In mouse white adipose tissue (SAT and VAT), lipid droplets were uneven in size, and the intercellular matrix was significantly increased. Large lipid droplets accumulated in the BAT of the experimental group mice, showing a trend towards whitening. Quantitative analysis of adipocyte size revealed abnormal volume distribution and reduced cell size uniformity in the white and brown adipocytes of the experimental group mice, suggesting an imbalance in adipose tissue homeostasis.

[0053] II. Construction of an Atherosclerosis Model in Adipose Tissue MED1-Specific Knockout Mice For the obtained MED1 ΔAdi Mice were injected with AAV8-PCSK9 virus via the tail vein and fed a high-cholesterol, high-fat diet to accelerate the development of severe atherosclerotic lesions. Phenotypic validation was performed on individuals exhibiting both significant characteristics of adipose malnutrition and atherosclerosis; these were designated as the adipose tissue MED1-specific knockout mouse model of atherosclerosis. The specific methods are as follows:

[0054] 1. Construction Steps MED1 gene knockout mice specifically targeting adipocytes (MED1) ΔAdi When the model mice reached 8 weeks of age, they were injected once via tail vein with 1×10 12 VG AAV8-PCSK9 virus (diluted to 10 with sterile saline) 13 Mice were injected with a dose of VG / mL (100 µL per mouse), followed by a high-cholesterol diet (purchased from Research Diets, D12109C, 40% fat, 1.25% cholesterol, 0.5% bile acids) for 12 weeks. This established a MED1-specific knockout mouse model of atherosclerosis in adipose tissue. Mice were euthanized at the end of the experiment to assess the severity of atherosclerosis.

[0055] 2. Evaluation of the MED1-specific knockout mouse model of atherosclerosis in adipose tissue (1) High-cholesterol feeding of MED1 at 0 months, 1 month, 2 months and 3 months fl / fl and MED1 ΔAdi Mice were anesthetized after fasting, blood samples were collected, and serum was separated. Triglyceride (TG) and total cholesterol (TC) levels were measured using a fully automated analyzer. Results are as follows: Figure 5 A~ Figure 5 As shown in Figure B, after tail vein injection of AAV8-PCSK9 virus to induce PCSK9 overexpression, the serum TC level in the adipose tissue MED1-specific knockout mouse atherosclerosis model (combined model group mice) was significantly increased.

[0056] (2) 8-week-old male MED1 fl / fl and MED1 ΔAdi Mice injected with AAV8-PCSK9 via the tail vein and fed a high-cholesterol diet for 12 weeks underwent heart and aortic perfusion treatment. The intact aorta was then removed and stained with Oil Red O to observe the overall plaque distribution. Results are as follows: Figure 6As shown, the aorta of the MED1-specific knockout mouse model of atherosclerosis showed obvious plaques, with plaque deposits in the aortic arch, thoracic aorta, and abdominal aorta.

[0057] (3) Take 8-week-old male MED1 mice fl / fl and MED1 ΔAdi Aortic root tissue from mice injected with AAV8-PCSK9 via the tail vein and fed a high-cholesterol diet for 12 weeks was prepared into frozen sections after OCT embedding. Oil Red O staining (to visualize lipid plaques) and HE staining (to observe plaque morphology and structure) were performed, and plaque area was quantitatively analyzed using image analysis software. Oil Red O staining and quantitative analysis of aortic root sections from model mice showed that the combined model group mice developed severe atherosclerotic lesions. Figure 7 ).

[0058] (4) After euthanasia, the animals were dissected, and the liver and three types of adipose tissue (SAT, VAT, BAT) were weighed. The results are as follows: Figure 8 As shown in Figure A, there were no significant differences in body weight between the two groups of mice before PCSK9 overexpression and at 1, 2, and 3 months after modeling. With increasing age and duration of high-fat diet intervention, MED1... ΔAdi Both the mice and their control mice showed an increase in body weight. However, MED1 ΔAdi The weight of the three types of adipose tissue (SAT, VAT, BAT) in mice compared to MED1 fl / fl The number of mice decreased significantly ( Figure 8 (B), liver weight and liver weight ratio were both significantly increased ( Figure 8 (C).

[0059] (5) H&E staining of adipose tissue sections showed that MED1 ΔAdi The number of lipid droplets in the white adipose tissue (SAT and VAT) of mice was significantly reduced, the interstitial matrix of adipocytes was increased, and a large number of small lipid droplets were observed; while in BAT, a large number of abnormally enlarged lipid droplets were observed. Figure 9 CD68, as a specific marker of macrophages, can be used to assess the level of inflammation. Immunohistochemical staining was used to analyze CD68 expression in adipose tissue of model mice. Figure 9 (B), the results showed MED1 ΔAdi The area of ​​CD68-positive tissue in mouse adipose tissue was significantly increased ( Figure 9 The C value indicates that the inflammatory response is significantly enhanced.

[0060] III. Application of a mouse model of lipodystrophy complicated with atherosclerosis in screening drugs for the prevention or treatment of lipodystrophy complicated with atherosclerosis. 1. Laboratory animals The adipose tissue MED1-specific knockout mouse model of atherosclerosis obtained in this invention was selected as the experimental group; MED1 fl / fl Mice were injected with AAV8-PCSK9 virus via the tail vein and fed a high-cholesterol, high-fat diet as a control group.

[0061] The candidate drugs were administered at different doses as needed and grouped into groups of at least 10 mice each. The experimental group mice and the control group mice were treated with different doses of different candidate drugs.

[0062] 2. Dosing strategy (1) The drug administration strategy for screening drugs to prevent lipodystrophy and atherosclerosis was to administer the drug at the same time as the start of tail vein injection of AAV8-PCSK9 virus and feeding of high cholesterol diet, and to evaluate the drug’s ability to prevent disease.

[0063] (2) The drug administration strategy for screening drugs to treat lipodystrophy complicated with atherosclerosis is as follows: AAV8-PCSK9 virus is injected into the tail vein and the diet is fed with high cholesterol for 4 weeks, and the drug administration is started and continued for 8 to 12 weeks.

[0064] 3. Taking the drug administration strategy for screening drugs to treat lipodystrophy complicated with atherosclerosis as an example, select specific routes of administration and indicator monitoring methods. The specific monitoring standards are as follows: (1) Route of administration: Based on the pharmacokinetic properties of the candidate drug, choose oral gavage, intraperitoneal injection or mixing into feed / drinking water.

[0065] (2) Dynamic monitoring indicators a. Weight: Record once a week.

[0066] b. Body composition analysis: Lean meat mass and fat mass were measured using a nuclear magnetic resonance body composition analyzer at the beginning, middle and end of the experiment.

[0067] c. Metabolic parameters Fasting blood glucose and serum insulin: every 4 weeks, HOMA-IR index was calculated to assess insulin resistance. Oral glucose tolerance test and insulin tolerance test: performed before drug administration and at the end of the test. Serum lipid profile: total cholesterol, triglycerides, LDL-C, and HDL-C were measured by tail blood sampling every 4 weeks.

[0068] 4. Sample collection and pharmacodynamic evaluation (1) Functional and morphological evaluation of adipose tissue Tissue weight: Accurately weigh and record the absolute weight and relative weight of subcutaneous fat, visceral fat, and brown fat. Histology and immunohistochemistry: H&E staining: Observe the size, uniformity of lipid droplet distribution, and intercellular matrix in each adipose tissue. Assess whether the drug improves phenotypes such as lipomatosis and BAT whitening in MED1ΔAdi mice. CD68 immunohistochemistry: Quantitatively analyze the infiltration of macrophages in adipose tissue and assess the inhibitory effect of the drug on adipose tissue inflammation.

[0069] Molecular biological analysis RT-qPCR / Western Blot: Detected the upregulation of expression of key genes for adipose function such as adiponectin and PPARγ, and the decrease in expression of inflammatory factors such as TNF-α and IL-6 in adipose tissue.

[0070] (2) Evaluation of atherosclerotic lesions Global plaque burden analysis of the aorta: Methods: The entire aorta was prepared and stained with Oil Red O.

[0071] Analysis: Image analysis software was used to quantify the percentage of plaque area in the entire aorta (from the aortic arch to the iliac bifurcation). This is the global gold standard for evaluating the efficacy of drugs in treating atherosclerosis.

[0072] Quantitative and stability analysis of aortic root plaques: Methods: Frozen sections were prepared from the aortic root and subjected to a series of staining.

[0073] Oil Red O staining: quantifies total plaque area and lipid core size.

[0074] Movat's five-color staining: analyzes plaque composition and assesses fibrous cap thickness and collagen content. Effective drugs should increase collagen and thicken the fibrous cap.

[0075] Immunofluorescence: Detect the co-localization of α-SMA (smooth muscle cell marker, which promotes stability) and CD68 (macrophage marker, which leads to instability), calculate the ratio of the two, and assess plaque stability.

[0076] (3) Evaluation of systemic metabolism and liver phenotype Serological analysis: Detection of adiponectin and leptin levels to assess fat function. Detection of inflammatory factors such as TNF-α, IL-6, and MCP-1 levels.

[0077] Liver analysis: Weighing: Calculate the liver weight / body weight ratio. H&E staining and Oil Red O staining: Assess the degree of hepatic steatosis (fatty liver). Hepatic triglyceride content measurement: Quantitatively analyze hepatic lipid deposition.

[0078] (4) Data analysis and efficacy determination Statistical analysis: All data are expressed as mean ± standard error. One-way ANOVA was used for comparisons among multiple groups. If variances were homogeneous, Tukey's post-hoc test was used for pairwise comparisons. A p-value < 0.05 was considered statistically significant.

[0079] Comprehensive criteria for evaluating drug efficacy: Main efficacy: Compared with the control group, the candidate drug group significantly reduced the overall plaque burden of the aorta (p<0.05) and / or significantly increased the stability indicators of aortic root plaques.

[0080] Secondary effectiveness: At least two of the following improvements are observed simultaneously: a. Improvement in adipose tissue weight and morphology (e.g., reduction of fat atrophy, reversal of BAT whitening).

[0081] b. Adipose tissue inflammation was significantly reduced (CD68+ area decreased, and expression of inflammatory factors decreased).

[0082] c. Increased systemic insulin sensitivity (decreased HOMA-IR, improved OGTT / ITT).

[0083] d. Improved lipid profile (especially reductions in LDL-C and TG).

[0084] e. Serum adiponectin levels were significantly elevated. Hepatic steatosis was reduced.

[0085] Candidate drugs that meet the primary efficacy criteria and are accompanied by relevant secondary efficacy indicators are considered to have the potential to treat lipodystrophy complicated with atherosclerosis in this model and are worthy of proceeding to the next stage of research and development.

[0086] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0087] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for constructing a mouse model of lipodystrophy complicated with atherosclerosis, characterized in that, Includes the following steps: MED1 flox / flox Mice and Adipoq-CreER T2 Mice were mated to obtain the MED1 genotype. fl / + / Adipoq-Cre + / - F1 generation mice; F1 generation mice were compared with MED1 flox / flox Backcrossing mice yielded F2 generation mice, with the MED1 genotype being [missing information]. fl / fl / Adipoq-Cre + / - Conditional knockout of the MED1 gene was achieved in mice induced by tamoxifen, resulting in an adipocyte-specific MED1 gene knockout mouse model. The adipocyte-specific MED1 gene knockout mouse model was obtained by injecting AAV8-PCSK9 virus into the tail vein and feeding it a high-cholesterol diet, thus creating a mouse model of lipodystrophy and atherosclerosis.

2. The method for constructing a mouse model of lipodystrophy complicated with atherosclerosis according to claim 1, characterized in that, Before tamoxifen induction, mice with the MED1 genotype from the F2 generation were used. fl / fl / Adipoq-Cre + / - Mice and genotype MED1 fl / fl / Adipoq-Cre - / - Mice were paired and bred to establish a stable genetic breeding line. Half of the offspring mice in this stable genetic breeding line were of genotype MED1. fl / fl / Adipoq-Cre + / - The mice were used as tamoxifen-induced experimental mice.

3. The method for constructing a mouse model of lipodystrophy complicated with atherosclerosis according to claim 1, characterized in that, The tamoxifen induction method is administered via intraperitoneal injection at a dose of 25 mg / kg body weight to 50 mg / kg body weight.

4. The method for constructing a mouse model of lipodystrophy complicated with atherosclerosis according to claim 1, characterized in that, The tail vein injection of AAV8-PCSK9 virus was 1×10⁻⁶. 12 VG~1×10 13 VG.

5. The method for constructing a mouse model of lipodystrophy complicated with atherosclerosis according to claim 1, characterized in that, Feed the animal with a high-cholesterol diet for 12 to 14 weeks.

6. The application of a mouse model of lipodystrophy complicated with atherosclerosis in screening drugs for the prevention or treatment of lipodystrophy complicated with atherosclerosis, characterized in that, The mouse model of lipodystrophy complicated with atherosclerosis was constructed by the method described in any one of claims 1 to 5.

7. The application according to claim 6, characterized in that, The application involves administering candidate drugs during the construction of a mouse model of lipodystrophy complicated with atherosclerosis, and screening for candidate drugs that can simultaneously improve fat function, correct metabolic disorders, and inhibit or reverse atherosclerotic plaques.

8. The application according to claim 7, characterized in that, The administration strategy for screening drugs to prevent lipodystrophy complicated with atherosclerosis was as follows: administration was performed simultaneously with the initiation of tail vein injection of AAV8-PCSK9 virus and feeding of a high-cholesterol diet, and the drug's ability to prevent disease was evaluated.

9. The application according to claim 7, characterized in that, The administration strategy for screening drugs to treat lipodystrophy complicated with atherosclerosis was as follows: AAV8-PCSK9 virus was injected via tail vein and the patient was fed a high-cholesterol diet for 4 weeks before administration began, and continued for 8 to 12 weeks.

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