ALD-related liver fibrosis mouse model mediated by AAV9-saCas9-sgYAP and its construction method

Abstract

The present invention discloses an ALD-related liver fibrosis mouse model mediated by AAV9-saCas9-sgYAP and a construction method thereof, wherein the construction method comprises the following steps: knocking out mouse Yes-associated protein 1 to obtain female YAP ‑ / ‑ Mice; female YAP mice were fed a Gao-binge alcohol diet. ‑ / ‑ The mice were raised and then gavaged with alcohol after the raising period. The construction method of the present invention is short in time and simple in operation, and can induce more severe liver fibrosis, providing a basis for studying the pathological mechanism of ALD in the middle and late stages.

Description

Technical Field

[0001] The present invention relates to the technical field of mouse models, and specifically to an ALD-related liver fibrosis mouse model mediated by AAV9-saCas9-sgYAP and a construction method. Background Art

[0002] Approximately 2 million people die from liver disease each year worldwide, and up to 50% of deaths from cirrhosis are attributed to the effects of alcohol. Alcohol-related liver disease (ALD) is one of the leading causes of death worldwide. ALD encompasses a range of pathological changes, including fatty liver, alcoholic hepatitis, liver fibrosis, and even cirrhosis. Although the severity of steatosis is an independent predictor of fibrosis progression, alcoholic hepatitis is indeed a high-risk factor for the progression of liver fibrosis. Approximately 3% of hepatitis patients develop cirrhosis each year. This also suggests that alcoholic hepatitis and liver fibrosis are key stages in the development of cirrhosis in ALD patients. Therefore, this stage is an ideal target for ALD treatment. However, due to the limitations of ALD models, the molecular mechanisms of alcoholic hepatitis and liver fibrosis remain to be elucidated.

[0003] The Gao-binge alcohol diet is widely used in ALD modeling, as it not only reproduces human drinking behavior but also closely mimics the pathological states of ALD, such as hepatic steatosis and mild inflammation. However, the ALD pathological mechanisms studied using this model remain limited to the early stages of ALD, lacking understanding of the pathological characteristics of ALD in key stages, such as hepatitis and liver fibrosis in the middle and late stages. Therefore, the construction of ALD hepatitis and liver fibrosis models is essential for exploring the pathological mechanisms of ALD in the middle and late stages.

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

[0005] The purpose of the present invention is to provide an ALD-related liver fibrosis mouse model mediated by AAV9-saCas9-sgYAP and a construction method. This construction method uses AAV9 to deliver the saCas9-sgYAP system (AAV9-saCas9-sgYAP) to perform gene editing on the mouse Yes-associated protein 1 gene to obtain mice with YAP gene deletion. On this basis, the ALD liver fibrosis model is successfully constructed by inducing a Gao-binge alcohol diet. This construction method is short in time, simple to operate, and can induce more severe liver fibrosis, providing a basis for studying the pathological mechanism of ALD in the middle and late stages.

[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 liver fibrosis mouse model mediated by AAV9-saCas9-sgYAP, the method comprising the following steps:

[0008] (a) Knockout of mouse Yes-associated protein 1 to obtain female YAP - / - mice (also known as AAV9-saCas9-sgYAP mice);

[0009] (b) Female YAP cells were fed the Lieber-DeCarli alcohol diet method. - / - The mice were raised, and after the raising period, alcohol was administered orally to obtain the ALD-related liver fibrosis mouse model.

[0010] Preferably, in step (a), knocking out mouse Yes-associated protein 1 comprises:

[0011] Screening for the sgRNA of the Yes-associated protein 1 gene, denoted as sgYAP, constructing a gene vector containing saCas9 and sgYAP, and then viral packaging to obtain AAV9-saCas9-sgYAP;

[0012] AAV9-saCas9-sgYAP was injected into mice via tail vein injection and female YAP cells were obtained after 1 to 3 weeks of feeding. - / - mouse.

[0013] Preferably, in step (b), the Gao-binge alcohol diet method comprises:

[0014] 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.

[0015] Preferably, the adaptive feeding time is 3 to 8 days, preferably 5 days.

[0016] Preferably, the continued feeding time is not less than 10 days.

[0017] Preferably, the continued feeding time is 10 days.

[0018] Preferably, the female YAP ΔHep The mice were 10 to 12 weeks old.

[0019] Preferably, in step (b), the number of gavages is 1, and the amount of alcohol gavage is 4-6 g / kg.

[0020] The second aspect of the present invention provides an ALD-related liver fibrosis mouse model constructed by the above-mentioned construction method and mediated by AAV9-saCas9-sgYAP.

[0021] Compared with the prior art, the beneficial effects of the present invention include at least:

[0022] The construction method of the present invention utilizes AAV9 to deliver the saCas9-sgYAP system (AAV9-saCas9-sgYAP) to perform gene editing on the mouse Yes-associated protein 1 gene, obtaining mice with YAP gene deletion. On this basis, a Gao-binge alcohol diet was used to induce an AAV9-saCas9-sgYAP-mediated ALD-related liver fibrosis mouse model. This construction method is time-saving, simple to operate, and can induce more severe liver fibrosis, providing a basis for studying the pathological mechanism of ALD in the middle and late stages. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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.

[0024] Figure 1 Western Blotting results of YAP protein in the liver of AAV9-saCas9-sgYAP mice;

[0025] Figure 2 The results of immunofluorescence detection of YAP protein in liver tissue of AAV9-saCas9-sgYAP mice;

[0026] Figure 3 Oil red "O" staining of liver sections from AAV9-saCas9-sgYAP mice fed a high-binge alcohol diet;

[0027] Figure 4 CD68 immunofluorescence staining of liver sections of AAV9-saCas9-sgYAP mice fed with a high-binge alcohol diet;

[0028] Figure 5 MPO immunofluorescence staining of liver sections of AAV9-saCas9-sgYAP mice fed a high-binge alcohol diet;

[0029] Figure 6Picrosirius red staining of liver sections from AAV9-saCas9-sgYAP mice fed a high-binge alcohol diet;

[0030] Figure 7 SMA immunofluorescence staining of liver sections from AAV9-saCas9-sgYAP 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 raw materials used in the following examples are as follows:

[0034] Lieber-DeCarli liquid feed was purchased from Research Diets, product number: L10016A.

[0035] Example 1

[0036] This example provides a method for constructing an ALD-related liver fibrosis mouse model mediated by AAV9-saCas9-sgYAP, which includes the following steps:

[0037] (a) Knockout of mouse Yes-associated protein 1 to obtain female YAP - / - mice;

[0038] Specifically, the researchers screened for the Yes-associated protein 1 gene, designated sgYAP, whose sequence is 5'-TGCTGGCAGTGGTACATCATC-3' (knockout is the second exon of the YAP gene), constructed a gene vector containing saCas9 and sgYAP, and then packaged the vector into AAV9-saCas9-sgYAP (provided by Shandong Weizhen Biological Co., Ltd.).

[0039] AAV9-saCas9-sgYAP (1×10 11 VG) were injected into mice and raised for 2 weeks to obtain female YAP - / - mice (also known as AAV9-saCas9-sgYAP mice);

[0040] Western Blotting to detect the expression of YAP protein in hepatocytes:

[0041] Liver tissue was isolated 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 results showed that YAP was highly expressed in wild-type mice, while only weak expression was observed in AAV9-saCas9-sgYAP mice ( Figure 1 ).

[0042] Depend on Figure 1 It can be seen that YAP is highly expressed in wild-type mice, while no YAP expression is observed in AAV9-saCas9-sgYAP mice;

[0043] 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 2 As shown;

[0044] Depend on Figure 2 It can be seen that a strong red fluorescent signal was observed in the liver of wild-type mice, indicating that the YAP protein was highly expressed in the liver, while no red fluorescent signal was observed in the liver of AAV9-saCas9-sgYAP mice, indicating that the YAP protein was missing in the liver;

[0045] (b) AAV9-saCas9-sgYAP mice were fed with Lieber-DeCarli fluid diet for 5 days;

[0046] (c) Female AAV9-saCas9-sgYAP mice were fed with Lieber-DeCarli alcohol fluid feed (5% alcohol) for 10 days. After the end of feeding, they were gavaged with alcohol at 5 g / kg body weight once to obtain an AAV9-saCas9-sgYAP-mediated ALD-related liver fibrosis mouse model.

[0047] Example 2

[0048] This example is a validation of the ALD-related liver fibrosis mouse model mediated by AAV9-saCas9-sgYAP in Example 1:

[0049] The animals were sacrificed 8 hours after gavage in step (c) of the method of Example 1, and samples were collected for testing;

[0050] 1) Oil red "O" staining of liver frozen sections

[0051] (1) Preparation of Oil Red "O"

[0052] 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.

[0053] 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.

[0054] (2) Oil red "O" staining of liver frozen sections

[0055] 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.

[0056] b. Immerse the sections in Oil Red "O" working solution for 30 minutes, then rinse with deionized water three times, 5 minutes each time.

[0057] c. Counterstain with hematoxylin for 6 minutes, then gently rinse with tap water for 5 minutes;

[0058] d. Glycerol gelatin sealing;

[0059] e. Microscope for image acquisition.

[0060] The collection results are as follows Figure 3 shown by Figure 3 It can be seen that after 15 days of feeding with a Gao-Binge alcohol diet, weak lipid accumulation appeared in the liver cells of wild-type mice, while lipid accumulation appeared in the liver cells of AAV9-saCas9-sgYAP mice, the lipid droplets increased significantly, and the liver fatty degeneration was obvious.

[0061] 2) Liver tissue was obtained for OCT embedding, sectioning (thickness: 7 μm), and fixation, and then immunofluorescence staining for CD68 and MPO was performed.

[0062] 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.

[0063] The results are as follows Figure 4 and 5 shown by Figure 4 and 5 It can be seen that after 15 days of feeding with Gao-Binge alcohol diet, the liver macrophages of wild-type mice ( Figure 4 ) and neutrophils ( Figure 5 ) infiltration was less, while macrophages ( Figure 4 ) and neutrophils ( Figure 5 ) Increased infiltration.

[0064] 3) Liver tissue was obtained for OCT embedding, sectioning (thickness: 7 μm), fixation, and Sirius red staining;

[0065] 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.

[0066] The results are as follows Figure 6 shown by Figure 6 It can be seen that after 15 days of feeding with a Gao-Binge alcohol diet, the degree of liver fibrosis in wild-type mice was mild, while the degree of liver fibrosis in AAV9-saCas9-sgYAP mice was aggravated.

[0067] 4) Liver tissue was obtained for OCT embedding, sectioning (thickness: 7 μm), and fixation, and SMA immunofluorescence staining was performed;

[0068] 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 SMA 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.

[0069] The results are as follows Figure 7 shown by Figure 7 It can be seen that after 15 days of feeding with the Gao-Binge alcohol diet, the wild-type mice expressed less α-SMA protein, while the AAV9-saCas9-sgYAP mice expressed increased α-SMA protein, mostly around the central vein.

[0070] 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 liver fibrosis mouse model mediated by AAV9-saCas9-sgYAP, characterized in that: The construction method comprises the following steps: (a) Knockout of Yes-associated protein 1 in mice resulted in female YAP - / - mice; (b) Female YAP cells were fed a Gao-binge alcohol diet. - / - The mice were raised, and after the raising period, alcohol was administered orally to obtain the ALD-related liver fibrosis mouse model; In the step (a), knocking out mouse Yes-associated protein 1 comprises: Screening for the sgRNA of the Yes-associated protein 1 gene, denoted as sgYAP, constructing a gene vector containing saCas9 and sgYAP, and then viral packaging to obtain AAV9-saCas9-sgYAP; AAV9-saCas9-sgYAP was injected into mice via tail vein injection. After 1-3 weeks of feeding, female YAP - / - mice; 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 female 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 ALD-related liver fibrosis mouse model constructed by the construction method according to any one of claims 1 to 3, mediated by AAV9-saCas9-sgYAP.

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