AAV9-CMV-Cre-mediated ALD-related hepatitis and liver fibrosis mouse model and construction method

Abstract

The present invention discloses an AAV9-CMV-Cre-mediated ALD-related hepatitis and liver fibrosis mouse model and a construction method thereof, wherein the construction method comprises: introducing YAP into female homozygous mice flox / flox In vivo injection of AAV9-CMV-cre adeno-associated virus and culture to obtain YAP ‑ / ‑ Mice; YAP was expressed in the Gao-binge alcohol diet. ‑ / ‑ The mice were raised and then gavaged with alcohol. This construction method successfully established an ALD-related hepatitis and liver fibrosis model in a short time and was able to induce more severe hepatitis and fibrosis, providing a basis for studying the late pathological mechanism of ALD.

Description

Technical Field

[0001] The present invention relates to the technical field of mouse models, and in particular to an AAV9-CMV-Cre-mediated ALD-related hepatitis and liver fibrosis mouse model and a construction method. Background Art

[0002] Alcohol-related liver disease (ALD) is the most common liver disease worldwide. ALD is not only the leading cause of liver-related deaths, but also the main indication for liver transplantation, accounting for 40% to 50% of all liver transplants each year. Approximately 10%-20% of ALD patients can develop cirrhosis. Steatosis, alcoholic hepatitis, and fibrosis are all closely related to the progression of cirrhosis. Among them, alcoholic hepatitis is a high-risk factor for cirrhosis, mainly due to its ability to accelerate the progression of liver fibrosis. Therefore, therapeutic strategies targeting inflammation and fibrosis may be an ideal way to reduce the mortality rate of ALD. However, due to the lack of models simulating ALD-related inflammation and fibrosis, the detailed mechanisms of these pathological changes remain unelucidated. In summary, the development of ALD-related inflammation and fibrosis models is of great significance for understanding the pathogenesis of ALD and exploring its treatment methods.

[0003] The high-binge alcohol diet is widely used to develop ALD mouse models. However, this model primarily mimics the pathological features of ALD, such as steatosis and mild inflammation. This means that a high-binge alcohol diet alone cannot replicate the inflammatory and fibrotic pathological changes associated with ALD.

[0004] In view of this, the present invention is proposed Summary of the Invention

[0005] The present invention aims to provide an AAV9-CMV-Cre-mediated ALD-related hepatitis and liver fibrosis mouse model and its construction method, wherein the construction method utilizes AAV9-CMV-cre adeno-associated virus to induce flox Yes-associated protein 1 (YAP) mice (YAP flox / flox ) to obtain YAP whole-body knockout mice (YAP - / - On this basis, an ALD-related hepatitis and liver fibrosis model was successfully established by feeding the mice with a Gao-binge alcohol diet. In addition, this construction method is time-consuming and can induce more severe hepatitis and fibrosis, providing a basis for studying the late pathological mechanism of ALD.

[0006] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0007] The first aspect of the present invention provides a method for constructing an ALD-related hepatitis and liver fibrosis mouse model mediated by AAV9-CMV-Cre, the method comprising the following steps:

[0008] (a) YAP expression in female homozygous mice flox / flox In vivo injection of AAV9-CMV-cre adeno-associated virus and culture to obtain YAP - / - mice;

[0009] (b) YAP according to the Lieber-DeCarli alcohol and diet method - / - The mice were raised, and after the raising period, they were gavaged with alcohol to obtain the AAV9-CMV-Cre-mediated ALD-related hepatitis and liver fibrosis mouse model.

[0010] Preferably, in step (a), homozygous mouse YAP Flox / Flox Loxp sites were inserted at both ends of the third exon of the YAP gene.

[0011] Preferably, in step (a), the feeding time is not less than 2 weeks.

[0012] Preferably, in step (b), the Gao-binge alcohol diet method comprises:

[0013] First, the fish were fed with Gao-binge fluid feed for adaptation, and then fed with Gao-binge fluid feed containing 5% alcohol for a period of time.

[0014] Preferably, the adaptive feeding time is 3 to 8 days; preferably, the feeding time is 5 days.

[0015] Preferably, the continued feeding time is not less than 10 days.

[0016] Preferably, the continued feeding time is 10 days.

[0017] Preferably, the YAP - / - The mice were 10 to 12 weeks old.

[0018] Preferably, in step (c), the number of gavages is 1, and the amount of alcohol gavage is 4-6 g / kg.

[0019] A second aspect of the present invention provides an AAV9-CMV-Cre-mediated ALD-related hepatitis and liver fibrosis mouse model constructed by the above-mentioned construction method.

[0020] Compared with the prior art, the beneficial effects of the present invention include at least:

[0021] The method of the present invention utilizes AAV9-CMV-cre adeno-associated virus to induce flox Yes associated protein 1 (YAP) mice (YAP flox / flox) to obtain YAP whole-body knockout mice (YAP - / - On this basis, an ALD-related hepatitis and liver fibrosis model was successfully established by feeding the mice with a Gao-binge alcohol diet. In addition, this construction method is time-consuming and can induce more severe hepatitis and fibrosis, providing a basis for studying the late pathological mechanism of ALD. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly describes the drawings required for the specific embodiments or the description of the prior art. Similar elements or parts are generally identified by similar reference numerals throughout the drawings. Elements or parts in the drawings are not necessarily drawn to scale.

[0023] Figure 1 For YAP flox / flox Mouse genotype detection electropherogram;

[0024] Figure 2 For YAP - / - Western Blotting results of YAP protein in mouse liver tissue;

[0025] Figure 3 For YAP - / - Results of immunofluorescence detection of YAP protein in mouse liver tissue;

[0026] Figure 4 For YAP - / - Oil red “O” staining of liver sections of mice fed with a Gao-binge alcohol diet;

[0027] Figure 5 For YAP - / - CD68 immunofluorescence staining of liver sections of mice fed with a high-binge alcohol diet;

[0028] Figure 6 For YAP - / - MPO immunofluorescence staining of liver sections of mice fed with a high-binge alcohol diet;

[0029] Figure 7 For YAP - / - Liver sections of mice fed with a Gao-binge alcohol diet were stained with picrosirius red;

[0030] Figure 8 For YAP - / - SMA immunofluorescence staining was performed on liver sections of mice fed a high-binge alcohol diet. DETAILED DESCRIPTION

[0031] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention and are therefore only used as examples and cannot be used to limit the scope of protection of the present invention.

[0032] It should be noted that, unless otherwise specified, the technical or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the present invention belongs.

[0033] The materials used in the following embodiments are as follows:

[0034] Homozygous YAP mice flox / flox :Obtained by the method in patent application number 202010654534.1 (for details, please refer to the specific implementation method);

[0035] AAV9-CMV-cre adeno-associated virus: provided by Shandong Weizhen Biological Co., Ltd.;

[0036] Lieber-DeCarli liquid feed was purchased from Research Diets, product number: L10016A.

[0037] Example 1

[0038] This example provides a method for constructing an ALD-related hepatitis and liver fibrosis mouse model mediated by AAV9-CMV-Cre, comprising the following steps:

[0039] (a) YAP expression in female homozygous mice flox / flox After in vivo injection of AAV9-CMV-cre adeno-associated virus and culturing for 2 weeks, YAP - / - mice;

[0040] 1. PCR-based YAP Flox / Flox Genotyping of mice

[0041] Flox Mouse Primers:

[0042] F1: 5'-GAACTCACATCTTTCCGGGTCCTG-3'

[0043] R1: 5'-TTCGGACCAGTCTGACGGATCC-3'

[0044] Flox homozygote: PCR product size is 351bp; Flox heterozygote: PCR product size is 351bp / 289bp; wild type: PCR product size is 289bp;

[0045] The PCR amplification reaction system is shown in Table 1:

[0046] Table 1

[0047]

[0048]

[0049] The PCR amplification reaction conditions are shown in Table 2:

[0050] Table 2

[0051]

[0052] Identification results such as Figure 1 As shown:

[0053] Depend on Figure 1 It can be seen that YAP flox / flox Homozygote: PCR product size is 351 bp; wild type: PCR product size is 289 bp;

[0054] 2. Detection of YAP using Western Blotting and Immunofluorescence Staining flox / flox and YAP - / - YAP expression in mouse liver

[0055] Liver tissue was separated and digested with protein lysis buffer to extract total protein. After denaturation with loading buffer, proteins were separated by SDS-PAGE electrophoresis and transferred to PVDF. Rabbit YAP antibody was incubated overnight at 4°C and HRP-labeled goat anti-rabbit IgG (H+L) was incubated at room temperature for 1 hour. YAP protein was detected by chemiluminescence. The detection results are as follows: Figure 2 As shown;

[0056] Depend on Figure 2 It can be seen that YAP flox / flox Mouse YAP protein is highly expressed, and YAP - / - No YAP protein expression was observed in mice;

[0057] Frozen sections (7 μm) of liver tissue were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100 at room temperature, blocked with 5% goat serum at room temperature for 1 hour, incubated with rabbit YAP antibody at 4°C overnight, incubated with Cy3-labeled goat anti-rat or rabbit secondary antibody at room temperature for 2 hours, stained with DAPI for 20 minutes, and mounted with antifade glycerol. The sections were observed and images were collected using a laser confocal microscope. Figure 3 As shown;

[0058] Depend on Figure 3 It can be seen that: YAP flox / floxA strong red fluorescent signal was observed in the mouse liver, indicating that YAP protein was highly expressed in the liver. - / - No red fluorescent signal was observed in the mouse liver, suggesting that YAP protein was missing in the liver.

[0059] (b) YAP was treated with Lieber-DeCarli fluid feed - / - Mice were housed for 5 days;

[0060] (c) YAP was treated with Lieber-DeCarli alcohol fluid diet (5% alcohol added) - / - The mice were raised for 10 days, and after the end of the raising period, 5 g / kg body weight of alcohol was administered orally once, thereby obtaining the AAV9-CMV-Cre-mediated ALD-related hepatitis and liver fibrosis mouse model.

[0061] Example 2

[0062] This example is a validation of the AAV9-CMV-Cre-mediated ALD-related hepatitis and liver fibrosis mouse model of Example 1:

[0063] The animals were sacrificed 8 hours after gavage in step (c) of the method of Example 1, and samples were collected for testing;

[0064] 1) Oil red "O" staining of liver frozen sections

[0065] (1) Preparation of Oil Red "O"

[0066] a. Dissolve 0.5 g of Oil Red "O" powder in a small amount of isopropanol. Then, dilute to 100 mL with isopropanol to prepare the storage solution. Store at 4°C in the dark.

[0067] b. When using, dilute the storage solution with deionized water in a ratio of 3:2, filter with qualitative filter paper, prepare the working solution, and use it up within 1 hour.

[0068] (2) Oil red "O" staining of liver frozen sections

[0069] a. Frozen liver sections were collected and rewarmed for 20 minutes. They were then fixed with 4% paraformaldehyde for 15 minutes and washed three times with deionized water, 5 minutes each time.

[0070] b. Immerse the sections in Oil Red "O" working solution for 30 minutes, then rinse with deionized water three times, 5 minutes each time.

[0071] c. Counterstain with hematoxylin for 6 minutes, then gently rinse with tap water for 5 minutes;

[0072] d. Glycerol gelatin sealing;

[0073] e. Microscope for image acquisition.

[0074] The collection results are as follows Figure 4 shown by Figure 4 It can be seen that after 15 days of Gao-Binge alcohol diet feeding, YAP flox / flox The mice showed subtle lipid accumulation in their liver cells, and YAP - / - Lipid accumulation appeared in the mouse liver cells, the number of lipid droplets increased significantly, and fatty degeneration of the liver was obvious.

[0075] 2) Liver tissue was obtained, embedded in OCT, sectioned (thickness: 7 μm), fixed, and immunofluorescence stained for CD68 (macrophage marker) and MPO (neutrophil marker);

[0076] Frozen sections of liver tissue (7 μm) were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100 at room temperature, blocked with 5% goat serum at room temperature for 1 hour, and then incubated with rat CD68 antibody and rabbit MPO antibody at 4°C overnight. They were then incubated with Cy3-labeled goat anti-rat or rabbit secondary antibodies at room temperature for 2 hours. Nuclei were stained with DAPI for 20 minutes, and the sections were mounted with antifade glycerol. The sections were observed and images were acquired using a laser confocal microscope.

[0077] The results are as follows Figure 5 and 6 shown by Figure 5 and 6 It can be seen that after 15 days of Gao-Binge alcohol diet feeding, YAP flox / flox Mouse liver macrophages ( Figure 5 ) and neutrophils ( Figure 6 ) infiltration is less, while YAP - / - Mouse liver macrophages ( Figure 5 ) and neutrophils ( Figure 6 ) Increased infiltration.

[0078] 3) Liver tissue was obtained for OCT embedding, sectioning (thickness: 7 μm), fixation, and Sirius red staining;

[0079] Frozen sections of liver tissue (7 μm) were equilibrated at room temperature for 10-15 min, fixed with 10% formalin at room temperature overnight, rinsed with running water, dried, stained with picrosirius red solution for 1 h, rinsed under running water for 6 min, stained with hematoxylin solution for 2 min, washed with running water for 6 min, dehydrated in a gradient manner with 80% ethanol, 90% ethanol, 95% ethanol, and 100% ethanol, made transparent with xylene, and sealed with neutral resin. Images were collected and analyzed under a microscope.

[0080] The results are as follows Figure 7 shown by Figure 7It can be seen that after 15 days of Gao-Binge alcohol diet feeding, YAP flox / flox The mice had milder liver fibrosis, and YAP - / - The degree of liver fibrosis in mice was aggravated.

[0081] 4) Liver tissue was obtained for OCT embedding, sectioning (thickness: 7 μm), and fixation, and immunofluorescence staining for α-actin (a marker of hepatic stellate cells) was performed.

[0082] Frozen sections of liver tissue (7 μm) were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100 at room temperature, blocked with 5% goat serum at room temperature for 1 hour, incubated with Cy3-labeled α-actin at room temperature for 2 hours, stained with DAPI for 20 minutes, and mounted with glycerol for observation and image acquisition using a laser confocal microscope.

[0083] The results are as follows Figure 8 shown by Figure 8 It can be seen that after 15 days of Gao-Binge alcohol diet feeding, YAP flox / flox The number of hepatic stellate cells in mouse livers is low, and YAP - / - There are a large number of hepatic stellate cells in the mouse liver, most of which are located around the central vein.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or make equivalent replacements for some or all of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and description of the present invention.

Claims

1. A method for constructing an ALD-related hepatitis and liver fibrosis mouse model mediated by AAV9-CMV-Cre, characterized in that: The construction method comprises the following steps: (a) YAP expression in female homozygous mice flox / flox In vivo injection of AAV9-CMV-cre adeno-associated virus and culture to obtain YAP - / - mice; (b) YAP according to Gao-binge alcohol and diet method - / - The mice were raised, and after the raising period, they were gavaged with alcohol to obtain the AAV9-CMV-Cre-mediated ALD-related hepatitis and liver fibrosis mouse model; In step (a), homozygous YAP mice flox / flox The third exon of the YAP gene has loxp sites inserted at both ends; In the step (b), the Gao-binge alcohol diet method comprises: First, the fish were fed with Gao-binge liquid feed for adaptation, and then fed with Gao-binge liquid feed containing 5% alcohol for a period of time; The adaptive breeding time is 3 to 8 days; The continued feeding time is 10 days.

2. The construction method according to claim 1, characterized in that The YAP - / - The mice were 10 to 12 weeks old.

3. The construction method according to claim 1, characterized in that In the step (b), the gavage frequency is 1 time, and the amount of alcohol administered is 4-6 g / kg.

4. An AAV9-CMV-Cre-mediated ALD-related hepatitis and liver fibrosis mouse model constructed by the construction method of any one of claims 1 to 3.

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