CN114223613A - Mouse model for hypercholesterolemia and atherosclerosis induced by AAV8-PCSK9 and construction method - Google Patents

Abstract

The invention discloses a mouse model of hypercholesterolemia and atherosclerosis based on AAV8-PCSK9 induction and a construction method, wherein the construction method comprises the following steps: homozygous mice yap1^Flox/FloxAnd Alb-Cre⁺Mice were crossed to obtain yap1^Flox/‑‑Alb‑Cre⁺F1 mouse of (1); selfing F1 mouse to obtain male yap1^Flox/Flox‑Alb‑cre⁺Mouse, notation yap1^ΔHepA mouse; to yap1^ΔHepInjecting AAV8-D377Y-mPCSK9 into mice; feeding the yak 1 injected with AAV8-D377Y-mPCSK9 with atherosclerosis feed^ΔHepAnd (3) mice, namely a mouse model of hypercholesterolemia and atherosclerosis induced by AAV8-PCSK 9. The construction method of the invention can form a simple hypercholesterolemia and atherosclerosis mouse model (namely that the plasma TC is high level, and the plasma TG is normal waterFlat); provides an ideal model for discussing the action mechanism of TG or other cardiovascular risk factors in atherosclerosis.

Description

Mouse model for hypercholesterolemia and atherosclerosis induced by AAV8-PCSK9 and construction method

Technical Field

The invention relates to the technical field of mouse models, in particular to a mouse model of hypercholesterolemia and atherosclerosis based on AAV8-PCSK9 induction and a construction method thereof.

Background

Simple hypercholesterolemia, elevated plasma total cholesterol (TC ≧ 190mg/dL), while plasma Triglycerides (TG) are at normal levels. Currently, apolipoprotein E knockout mice (ApoE)^-/-) Or low density lipoprotein receptor knockout mice (LDLR)^-/-) Is a classical gene modification model for atherosclerosis research. In addition, adeno-associated virus serotype 8(AAV8) -mediated overexpression of the proprotein convertase subtilisin-type 9 (PCSK9) (AAV8-PCSK9) is also a common method for inducing atherosclerosis in wild-type mice.

The atherosclerotic diet induced hypercholesterolemia in the above model accompanied by hypertriglyceridemia (TG > 150 mg/dL). This suggests that the above mouse model is only able to induce combined hyperlipidemia, not simple hypercholesterolemia. Due to the lack of animal models, the role that simple hypercholesterolemia plays in atherogenesis is unclear; therefore, it is necessary to construct a simple hypercholesterolemia model.

Disclosure of Invention

The invention aims to provide a mouse model of hypercholesterolemia and atherosclerosis induced by AAV8-PCSK9 and a construction method thereof, wherein the mouse model of simple hypercholesterolemia can be constructed by the construction method, namely TC is at a high level, and TG is at a normal level.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

the invention provides a construction method of a mouse model based on AAV8-PCSK9 induced hypercholesterolemia and atherosclerosis, which comprises the following steps:

(a) homozygous mice yap1^Flox/FloxAnd Alb-Cre⁺Mice were crossed to obtain yap1^Flox/--Alb-Cre⁺F1 mouse of (1); selfing F1 mouse to obtain male yap1^Flox/Flox-Alb-cre⁺Mouse, notation yap1^ΔHepA mouse;

(b) through the tail vein to yap1^ΔHepInjecting AAV8-D377Y-mPCSK9 into mice;

(c) feeding the yak 1 injected with AAV8-D377Y-mPCSK9 with atherosclerosis feed^ΔHepAnd (3) obtaining the mouse model of the hypercholesterolemia and the atherosclerosis based on the AAV8-PCSK9 induction.

In the present invention, the homozygous mouse yap1 in step (a) is treated^Flox/FloxThe construction method of (A) is not particularly restricted, and for example, a method conventionally employed in the art can be employed; preferably, the method is as described in patent application No. 202010654534.1 (see the detailed description).

Preferably, in said step (b), yap1^ΔHepThe mice are 2-6 weeks old.

Preferably, the injection amount of the AAV8-D377Y-mPCSK9 is 1 x 10¹¹vg/m.

Preferably, said homozygous mouse yap1^Flox/FloxThe 3 rd exon of yap1 gene has loxp sites inserted into both ends.

Preferably, said homozygous mouse yap1^Flox/FloxAnd Alb-Cre⁺The male-female ratio of the mouse is 1: 1-5.

Preferably, the ratio of male and female in the F1 generation mouse selfing is 1: 1-5.

Preferably, the feeding time is not less than 5 weeks.

Preferably, the feeding time is 12 weeks.

The second aspect of the invention provides a mouse model of the hypercholesterolemia and the atherosclerosis based on the AAV8-PCSK9 induction, which is constructed by the construction method.

Compared with the prior art, the invention has the beneficial effects that at least:

the invention constructs YAP1 gene (the gene codes for co-transcriptional activator: yes-related protein namely YAP) liver cell specific knockout mice (YAP 1)^ΔHep) On the basis, by single injection of AAV8(AAV8-D377Y-mPCSK9) which overexpresses PCSK9 gain-of-function mutant and atherosclerosis feed, a mouse model of simple hypercholesterolemia and atherosclerosis (namely plasma TC is high level, and plasma TG is normal level) is successfully induced; provides an ideal model for discussing the action mechanism of TG or other cardiovascular risk factors in atherosclerosis.

The construction method avoids YAP^ΔHepCompared with the classical gene modified atherosclerosis model (including ApoE)^-/-And LDLR^-/-Mice) in cage breeding work, which is beneficial to reducing the breeding cost and shortening the experimental period.

Drawings

In order to more clearly illustrate the detailed description of the invention or the technical solutions in the prior art, the drawings that are needed in the detailed description of the invention or the prior art will be briefly described below. Throughout the drawings, like elements or portions are generally identified by like reference numerals. In the drawings, elements or portions are not necessarily drawn to scale.

FIG. 1 shows yap1^ΔHepDetecting an electrophoretogram of the mouse genotype;

FIG. 2 shows yap1^ΔHepThe result of the mouse hepatocyte YAP protein western Blotting detection;

FIG. 3 shows yap1^ΔHepCarrying out immunofluorescence detection on the mouse hepatocyte YAP protein;

FIG. 4 shows Yap1 injected with AAV8-D377Y-mPCSK9^flox/floxMouse and yap1^ΔHepPlasma TC and TG levels in mice fed cholesterol diet for 12 weeks;

FIG. 5 shows Yap1 injected with AAV8-D377Y-mPCSK9^flox/floxMouse and yap1^ΔHepCholesterol feed for miceAfter 12 weeks of feeding, liver sections were stained for oil red "O" and liver TG levels;

FIG. 6 shows Yap1 injected with AAV8-D377Y-mPCSK9^flox/floxMouse and yap 1. delta^HepAfter the mice are fed with cholesterol feed for 12 weeks, aortic tree oil red 'O' staining and large atherosclerotic plaque area analysis are carried out;

FIG. 7 shows yap1 injected with AAV8-D377Y-mPCSK9^flox/floxMouse and yap1^ΔHepAfter the mice are fed with cholesterol feed for 12 weeks, carrying out HE and oil red O staining on aortic root cryosections and analyzing the area of the atherosclerotic plaques under the microscope;

FIG. 8 shows yap1 injected with AAV8-D377Y-mPCSK9^flox/floxMouse and yap1^ΔHepAfter the mice were fed with cholesterol feed for 12 weeks, immunofluorescence staining and plaque cell composition analysis of aortic root cryo-section CD68 (macrophage marker protein) and alpha-actin (vascular smooth muscle cell marker protein).

Detailed Description

The following describes embodiments of the present invention in detail with reference to the following embodiments. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.

It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains.

The following examples employ the following materials:

homozygous mouse yap1^Flox/Flox: obtained by adopting the method in patent application No. 202010654534.1 (see the specific embodiment);

Alb-Cre⁺mice: offered by seiko biotechnology limited;

atherosclerosis feed: supplied by Ruidi Biotech (Shenzhen) Limited (Cat: D12109C).

AAV8-D377Y-mPCSK 9: supplied by Shandong Weizhen Biotech, Inc.

Examples

This example is a method for constructing a mouse model based on AAV8-PCSK 9-induced hypercholesterolemia and atherosclerosis, comprising the steps of:

firstly, homozygous mice yap1 are mixed according to the male-female ratio of 1: 3^Flox/FloxAnd Alb-Cre⁺Mice were crossed to obtain yap1^Flox/--Alb-Cre⁺F1 mouse of (1); selfing F1 mouse to obtain male yap1^Flox/Flox-Alb-cre⁺Mouse, notation yap1^ΔHepThe male-female ratio in the inbred of the F1 mouse is 1: 3;

1. PCR for yap1^ΔHepMouse genotype identification

The specific identification method is as follows:

and (3) PCR identification:

flox mouse primers:

F：

5’-GAACTCACATCTTTCCGGGTCCTG-3’；

R：5’-TTCGGACCAGTCTGACGGATCC-3’；

the PCR amplification reaction system is shown in Table 1;

TABLE 1

The PCR amplification reaction conditions are shown in Table 2;

TABLE 2

Alb-cre mouse primers:

Alb-cre-F：

5’-GAAGCAGAAGCTTAGGAAGATGG-3’；

Alb-cre-R：

5’-TTGGCCCCTTACCATAACTG-3’；

the PCR amplification reaction system is shown in Table 3;

TABLE 3

The PCR amplification reaction conditions are shown in Table 4;

TABLE 4

The results of the electrophoretic analysis are shown in FIG. 1;

as can be seen from fig. 1: flox homozygote: the size of the PCR product is 351 bp; wild type: the PCR product size was 289 bp. In addition, when the primers Alb-cre-F and Alb-cre-R are amplified by PCR, the product containing Alb-cre gene is 390bp, and the wild type has no band.

Western Blotting detection of hepatocyte YAP protein expression

Separating liver tissues, digesting the tissues by using a protein lysate, extracting total protein, adding a sample loading buffer solution for denaturation, separating the protein by SDS-PAGE electrophoresis, transferring the protein on PVDF (polyvinylidene fluoride), incubating rabbit-derived YAP antibody at 4 ℃ overnight, incubating goat anti-rabbit IgG (H + L) marked by HRP (horse radish peroxidase) for 1 hour at room temperature, and detecting the YAP protein by using a chemiluminescence method, wherein the detection result is shown in figure 2;

as can be seen from fig. 2: wild type and yap1^flox/floxMouse YAP is in high expression state, while YAP1^ΔHepNo expression was observed. This indicates that yap1 gene was knocked out in liver tissue.

3. Immunofluorescence staining detection of hepatocyte YAP protein expression

Fixing a frozen section (7 mu m) of liver tissue by 4% paraformaldehyde for 15 minutes, permeabilizing 0.1% Triton X-100 at room temperature, sealing the frozen section by 5% goat serum at room temperature for 1 hour, respectively incubating with goat-derived albumin (Alb) (hepatocyte labeled protein) antibody and rabbit-derived YAP antibody at 4 ℃ overnight, incubating Cy 3-labeled donkey anti-goat and Alexa-Fluor 488-labeled donkey anti-rabbit antibody at room temperature for 2 hours, staining nuclei with DAPI for 20min, sealing an anti-quencher glycerol, observing by using a laser confocal microscope and collecting images, wherein the result is shown in figure 3;

as can be seen from fig. 3: wild type, yap1^flox/floxAnd yap1^ΔHepThe livers of the mice all show stronger red fluorescence signals, wild type and yap1^flox/floxThe strong green fluorescence signal in mouse liver indicates that YAP is in high expression state in liver, YAP is not knocked out, and YAP1^ΔHepNo green fluorescent signal was seen in the liver, suggesting that YAP had been successfully knocked out in hepatocytes.

Second, by tail vein injection, 4 weeks old yap1^flox/floxXiaohe yap1^ΔHepAAV8-D377Y-mPCSK9 is injected into mice in vivo, and the injection amount is 1 x 10¹¹vg/m;

thirdly, feeding the atherosclerosis feed with yap1 injected with AAV8-D377Y-mPCSK9^ΔHepMice were obtained for 12 weeks, and a simple hypercholesterolemia and atherosclerosis mouse model was obtained.

1. A mouse model was constructed as described in the above examples, yap1 at 0, 4, 8 and 12 weeks, respectively^ΔHepMouse and yap1^Flox/FloxAnd (3) detecting the TC and TG levels of the blood plasma of the mouse, wherein the detection method specifically comprises the following steps:

1) plasma harvesting

(1) The blood adopts heparin sodium as anticoagulant; before blood collection, adding anticoagulant (generally 20 mu L) into 0.2ml centrifuge tubes respectively, and drying for later use;

(2) the blood sampling part is the tail of a mouse; blood is collected by capillary after heparin sodium treatment.

(3) Placing the blood in a low temperature centrifuge, centrifuging at 3000rpm for 20min, collecting supernatant, and storing in a refrigerator at-80 deg.C.

2) Plasma TC detection

(1) The method comprises the following steps: the TC assay was performed by an enzymatic method.

(2) The principle is as follows:

(3) the method comprises the following steps:

a. preparation of standard product: the standard (200mg/dL) was taken out from a freezer at-20 ℃ and thawed for use.

b. Preparation of a standard curve: the diluted standards were added to 96-well plates in the proportions shown in table 5, respectively, and 3 multiple wells were set.

c. Dilution of plasma: all plasma samples were diluted as appropriate depending on the sample (note: the samples were kept as short as possible during the dilution).

d. Sample adding: the diluted samples were added to a 96-well plate, 20. mu.l, next to the standard curve wells, in 3 duplicate wells.

e. Adding a color developing reagent: 200. mu.L of the chromogenic working solution was added to each well.

Incubation at f.37 ℃ for 10 min.

g. The detection is carried out by a microplate reader, and the wavelength is 492 nm.

h. And calculating the concentration of the sample by using an equation of the standard curve according to the OD value of the sample. And finally, multiplying the obtained data by the dilution factor to obtain an original sample value.

TABLE 5 plasma TC assay kit dilution table

3) Plasma TG detection

(1) The method comprises the following steps: TG detection was performed by an enzymatic method.

(2) The principle is as follows:

(3) the method comprises the following steps:

a. preparation of standard product: the standard (200mg/dL) was taken out from a freezer at-20 ℃ and thawed for use.

b. Preparation of a standard curve: the diluted standards were added to 96-well plates in the proportions shown in table 6, respectively, and 3 multiple wells were set.

c. Dilution of plasma: all plasma samples were diluted as appropriate depending on the sample (note: the samples were kept as short as possible during the dilution).

d. Sample adding: the diluted sample was added to a 96-well plate, 20 μ L, next to the standard curve well, in 3 duplicate wells.

e. Adding a reagent: 200. mu.L of the chromogenic working solution was added to each well.

Incubation at f.37 ℃ for 10 min.

g. The detection is carried out by a microplate reader, and the wavelength is 492 nm.

h. And calculating the concentration of the sample by using an equation of the standard curve according to the OD value of the sample. And finally, multiplying the obtained data by the dilution factor to obtain an original sample value.

TABLE 6 dilution table of plasma TG detection kit

yap1^ΔHepMouse and yap1^Flox/FloxThe results of the measurement of plasma TC and TG levels of the mice are shown in fig. 4;

from the figure4, injection of AAV8-D377Y-mPCSK9 at yap1^flox/floxMouse and yap1^ΔHepAfter the mice were fed with the atherosclerotic diet, plasma TC levels were 811.57 + -37.61 mg/dL, 623.27 + -60.77 mg/dL, respectively, but plasma TG levels were 275 + -33.27 mg/dL, 135.99 + -25.17 mg/dL, respectively. This suggests that although yap1 knock-out of hepatocytes significantly reduced plasma TC levels, plasma TC remained at a higher level, i.e., hypercholesterolemia. However, yap1 knockdown of hepatocytes significantly reduced plasma TG levels and remained at normal levels. In conclusion, the atherosclerotic feed was able to induce yap1 injection of AAV8-D377Y-mPCSK9^ΔHepMice develop simple hypercholesterolemia.

2. Atherosclerosis feed yap1^flox/floxMouse and yap1^ΔHepAfter 12 weeks of mice, liver tissues were subjected to OCT embedding, sectioning (thickness: 7 μm), fixation and oil red "O" staining; another part of liver tissue was homogenized to detect TG levels in the liver.

1) Oil red 'O' staining of liver frozen sections

(1) Preparation of oil red

a. Dissolving 0.5 g of oil red O dry powder in a small amount of isopropanol, adding isopropanol to a constant volume of 100mL, preparing a storage solution, and storing at 4 ℃ in a dark place;

b. when the water-saving agent is used, the storage solution and deionized water are diluted according to the proportion of 3:2, qualitative filter paper is used for filtering, and working solution is prepared and used up within 1 hour.

(2) Oil red 'O' staining of liver frozen sections

a. Freezing liver, slicing, rewarming for 20min, fixing with 4% paraformaldehyde for 15min, washing with deionized water for 3 times and 5 min/time;

b. soaking the slices in oil red O working solution for 30min, and washing with deionized water for 3 times and 5 min/time;

c. counterstaining with hematoxylin for 6min, and gently cleaning with tap water for 5 min;

d. sealing the glycerol gelatin into pieces;

e. and (5) acquiring pictures by using a microscope.

2) Liver TG level detection

a. To the liver tissue, 9 times the volume of chloroform-methanol mixture (2:1) was added to homogenize the liver tissue, and a 10% homogenate was prepared. Centrifuging at 5000rpm for 15min, collecting supernatant, blowing with nitrogen gas, and dissolving with anhydrous ethanol.

b. And (3) carrying out TG level detection on the absolute ethanol complex solution by using the TG detection method.

The results are shown in FIG. 5; as can be seen from FIG. 5, yap1 was injected with AAV8-D377Y-mPCSK9^flox/floxThe mice showed massive lipid accumulation in hepatocytes 12 weeks after induction with atherosclerotic diet, whereas yap1^ΔHepOnly a weak accumulation of lipids occurred in hepatocytes of mice. The results of testing liver TG levels showed that yap1 was comparable to that of AAV8-D377Y-mPCSK9^flox/floxMouse comparison, yap1^ΔHepLiver TG levels in mice were significantly reduced and normal levels were maintained.

3. Mice fed with the atherosclerosis feed of the examples for 12 weeks were euthanized, the aortic trees of the mice were isolated under a stereomicroscope, and after 12 hours of fixation with 4% paraformaldehyde, oil red "O" staining was performed to count the area of the gross plaques. Separating the heart, carrying out OCT embedding, slicing (7 mu m), fixing, carrying out HE and oil red O staining, and observing the area of the plaque under a statistical microscope;

1) oil red "O" staining of mouse aortic trees;

fixing 4% paraformaldehyde for 12 hours, then placing the mouse aortic tree in oil red O working solution for dip dyeing for 2 hours, flushing gently for 5 minutes by running water, longitudinally splitting the aortic blood vessel by using Venus scissors, fixing the aortic blood vessel in a black rubber dish by using a fine needle, taking a picture, collecting the picture, and carrying out statistical analysis on the area of the plaque by using ipp6 software. The results are shown in FIG. 6. As shown in FIG. 6, after AAV8-D377Y-mPCSK9 was injected, the atherosclerosis feed induced yap1^flox/floxMouse and yap1^ΔHepMice developed atherosclerotic plaques. However, with yap1^flox/floxMouse comparison, yap1^ΔHepThe mouse aorta and aortic arch gross plaque area was significantly reduced.

2) Oil red "O" and HE staining of main cryosection of aortic root

(1) Oil red "O" staining

a. Taking a frozen section (7 mu m) of aortic sinus at the root of the heart, after rewarming for 20min, fixing for 15min by 4% paraformaldehyde, washing for 3 times by deionized water for 5 min/time;

b. soaking the slices in oil red O working solution for 30min, and washing with deionized water for 3 times and 5 min/time;

c. counterstaining with hematoxylin for 6min, and gently cleaning with tap water for 5 min;

d. sealing the glycerol gelatin into pieces;

e. and (5) acquiring pictures by using a microscope.

(2) HE staining

a. Taking a frozen section (7 mu m) of aortic sinus at the root of the heart, after rewarming for 20min, fixing for 15min by 4% paraformaldehyde, washing for 3 times by deionized water for 5 min/time;

b. the slices are placed in hematoxylin for dip-dyeing for 8min, and are lightly washed with tap water for 5 min;

c, differentiating by using 1% hydrochloric acid alcohol for 30sec, and slightly washing by using tap water for 5 min;

d. redyeing for 2 min;

e. placing the slices in 95% alcohol I1 min-95% alcohol II 1 min-absolute ethanol I1 min-absolute ethanol II 1 min-xylene I1 min-xylene II 1min for dehydration and transparency, taking out the slices from xylene, slightly drying, and sealing with neutral gum;

f. and (5) acquiring pictures by using a microscope.

Yap1 injected with AAV8-D377Y-mPCSK9^flox/floxMouse and yap1^ΔHepAfter 12 weeks of feeding the mice with the cholesterol diet, aortic root aortic sinus HE and oil red "O" staining and microscopic atherosclerotic plaque area analysis are shown in fig. 7.

As shown in FIG. 7, the injection of AAV8-D377Y-mPCSK9 induced yap1 in the atherogenic diet^{flox /flox}Mouse and yap1^ΔHepIn mice, obvious plaques appeared in the roots of the aorta under the scope of the endoscope, however, with yap1^flox/floxMouse comparison, yap1^ΔHepThe plaque area in the aortic root of the mice was significantly reduced.

3) Immunofluorescence staining of CD68 (macrophage marker protein molecule) and alpha-actin (SMA, vascular smooth muscle cell marker protein molecule) of heart aorta root frozen section

The method comprises the following steps of fixing a heart aorta root frozen section for 15 minutes by 4% paraformaldehyde, permeabilizing 0.1% Triton X-100 at room temperature, sealing the heart aorta root frozen section for 1 hour at room temperature by 5% goat serum, incubating the heart aorta root frozen section with rabbit-derived CD68 antibody at 4 ℃ overnight, incubating Cy 3-labeled mouse-derived alpha-actin and Alexa-Fluor 488-labeled goat anti-rabbit antibody for 2 hours at room temperature, staining a nucleus with DAPI for 20 minutes, sealing an anti-quencher glycerol, observing by using a laser confocal microscope and collecting images.

The results of immunofluorescent staining for CD68 and alpha-actin are shown in FIG. 8.

As can be seen from FIG. 8, yap1 was found for the cell component in plaque^flox/floxMouse comparison, yap1^ΔHepMice produced a significant reduction in the amount of macrophages in the plaques. However, there was no significant difference in vascular smooth muscle cell content within the plaques formed in the two mice.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present invention, and they should be construed as being included in the following claims and description.

Claims (9)

1. A method for constructing a mouse model based on AAV8-PCSK9 induced hypercholesterolemia and atherosclerosis is characterized by comprising the following steps:

(a) homozygous mice yap1^Flox/FloxAnd Alb-Cre⁺Mice were crossed to obtain yap1^Flox/--Alb-Cre⁺F1 mouse of (1); selfing F1 mouse to obtain male yap1^Flox/Flox-Alb-cre⁺Mouse, notation yap1^ΔHepA mouse;

(b) to yap1^ΔHepIn vivo injection of AAV8-D377Y-mP in miceCSK9；

(c) Feeding the yak 1 injected with AAV8-D377Y-mPCSK9 with atherosclerosis feed^ΔHepAnd (3) obtaining the mouse model of the hypercholesterolemia and the atherosclerosis based on the AAV8-PCSK9 induction.

2. The method of claim 1, wherein in step (b), yap1^ΔHepThe mice are 2-6 weeks old.

3. The method according to claim 1 or 2, wherein the AAV8-D377Y-mPCSK9 is injected in an amount of 1 x 10¹¹vg/m.

4. The method of claim 1, wherein said homozygous mouse yap1^Flox/FloxThe 3 rd exon of the Yap gene is inserted with loxp sites at two ends respectively.

5. The method of claim 1, wherein said homozygous mouse yap1^Flox/FloxAnd Alb-Cre⁺The male-female ratio of the mouse is 1: 1-5.

6. The construction method according to claim 1, wherein the ratio of male and female in the F1 generation mouse selfing is 1: 1-5.

7. The method of constructing according to claim 1, wherein the feeding time is not less than 5 weeks.

8. The method of constructing according to claim 1, wherein the feeding period is 12 weeks.

9. A mouse model of hypercholesterolemia and atherosclerosis induced by AAV8-PCSK9 and constructed by the construction method of any one of claims 1-8.

Images