An ald late-stage related liver fibrosis mouse model and a construction method thereof

Abstract

The application discloses an ALD-late-stage-related liver fibrosis mouse model and a construction method thereof, and the construction method comprises the following steps: crossing a homozygous mouse YAP flox / flox with an Alb-Cre + mouse to obtain F1 mice of YAP flox / ‑ -Alb-Cre + ; the F1 generation mice are self-crossed to obtain male YAP flox / flox -Alb-cre + mice, which are recorded as YAP ΔHep mice; the YAP ΔHep mice are bred according to a Gao-binge alcohol diet method, and alcohol is used for intragastrical administration after the breeding is completed; the construction method is used for constructing the YAP gene hepatocyte-specific knockout mouse YAP ΔHep on the basis of successfully constructing the ALD-late-stage-related liver fibrosis mouse model by using the Gao-binge alcohol diet induction; the construction method is short in time, the induced fibrosis is serious, and a basis is provided for studying the pathological mechanism of the ALD late stage.

Description

Technical Field

[0001] This invention relates to the field of mouse model technology, specifically to a mouse model of late-stage ALD-related liver fibrosis and its construction method. Background Technology

[0002] Alcohol-related liver disease (ALD) is a series of liver injuries caused by alcohol, with major pathological changes including steatosis, inflammation, fibrosis, and even liver cancer. Cirrhosis caused by ALD is one of the leading causes of death from liver disease. The pathological mechanisms of ALD are extremely complex and remain unclear. Currently, there are still no effective means for the prevention and treatment of ALD.

[0003] Because the Gao-binge alcoholic diet not only reproduces human drinking behavior but also highly simulates the pathological features of early-stage ALD, such as hepatic steatosis and inflammation, it has been widely used in ALD model creation. However, the pathological mechanisms of ALD studied using this model are limited to the early stages of ALD, lacking understanding of the pathological mechanisms of key stages (such as the middle and late stages). Liver fibrosis is an important pathological feature of middle and late-stage ALD; therefore, replicating an ALD liver fibrosis model is essential for exploring the pathological mechanisms of middle and late-stage ALD.

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

[0005] The purpose of this invention is to provide a mouse model of advanced ALD-related liver fibrosis and its construction method. This construction method is used to construct YAP gene hepatocyte-specific knockout mice. ΔHep Based on this, a mouse model of late-stage ALD-related liver fibrosis was successfully constructed using Gao-binge alcohol diet induction. This construction method is quick and induces severe fibrosis, providing a foundation for studying the pathological mechanisms of late-stage ALD.

[0006] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0007] The first aspect of this invention provides a method for constructing a mouse model of late-stage ALD-related liver fibrosis, the method comprising the following steps:

[0008] (a) YAP mice homozygous flox / flox With Alb-Cre + YAP was obtained by hybridizing mice flox / --Alb-Cre + F1 mice; F1 mice were self-crossed to obtain male YAP mice. flox / flox -Alb-cre + Mice, denoted as YAP ΔHep Mice;

[0009] (b) YAP was administered according to the Lieber-DeCarli alcohol diet method. ΔHep Mice were fed, and after the feeding period ended, they were given alcohol by gavage to obtain the mouse model of late-stage ALD-related liver fibrosis.

[0010] Preferably, in step (a), homozygous mice YAP flox / flox The third exon of the YAP gene has loxp sites inserted at both ends.

[0011] Preferably, in step (a), homozygous mice YAP flox / flox With Alb-Cre + The male-to-female ratio of mice was 1:(1-5).

[0012] Preferably, in step (a), the female-to-male ratio in the self-crossing of F1 generation mice is 1:(1-5).

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

[0014] First, acclimatize the animals with Gao-binge liquid feed, then continue feeding them with Gao-binge liquid feed containing 5% alcohol for a period of time.

[0015] Preferably, the adaptation period is 3 to 8 days; more preferably, it is 5 days.

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

[0017] Preferably, the YAP ΔHep The mice were 10–12 weeks old.

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

[0019] A second aspect of the present invention provides a mouse model of late-stage ALD-related liver fibrosis constructed by the above-described construction method.

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

[0021] The method of this invention is used to construct YAP gene hepatocyte-specific knockout mice (YAP). ΔHep Based on this, a mouse model of late-stage ALD-related liver fibrosis was successfully constructed using Gao-binge alcohol diet induction. This construction method is quick and induces severe fibrosis, providing a foundation for studying the pathological mechanisms of late-stage ALD. Attached Figure Description

[0022] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0023] Figure 1 YAP ΔHep Electrophoresis results of mouse genotyping;

[0024] Figure 2 YAP ΔHep Western blotting results of YAP protein detection and quantification in mouse liver tissue;

[0025] Figure 3 YAP ΔHep Immunofluorescence assay results of YAP protein in mouse hepatocytes;

[0026] Figure 4 YAP ΔHep Liver sections of mice fed a Gao-binge alcohol diet were stained with Oil Red "O" staining.

[0027] Figure 5 YAP ΔHep After mice were fed a Gao-binge alcohol diet, liver sections were stained with CD68 immunofluorescence.

[0028] Figure 6 YAP ΔHep Mice were fed a Gao-binge alcohol diet, and liver sections were stained with MPO immunofluorescence.

[0029] Figure 7 YAP ΔHep Liver sections of mice fed a Gao-binge alcohol diet were stained with Sirius red.

[0030] Figure 8 YAP ΔHep After mice were fed a Gao-binge alcohol diet, liver sections were stained with α-actin immunofluorescence. Detailed Implementation

[0031] The embodiments of the technical solution of the present invention will be described in detail below with reference to the examples. The following embodiments are only used to illustrate the technical solution of the present invention more clearly, and are therefore only examples, and should not be used to limit the scope of protection of the present invention.

[0032] It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning as understood by those skilled in the art to which this invention pertains.

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

[0034] homozygous mice YAP flox / flox The method described in patent application number 202010654534.1 is used (see detailed implementation details for specific information).

[0035] Alb-Cre + Mice: Provided by Cyagen Biosciences Co., Ltd.

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

[0037] Example 1

[0038] This embodiment describes a method for constructing a mouse model of late-stage ALD-related liver fibrosis, which includes the following steps:

[0039] (a) Homozygous mice YAP were divided into male and female groups at a ratio of 1:3. flox / flox With Alb-Cre + YAP was obtained by hybridizing mice flox / --Alb-Cre + F1 mice; F1 mice were self-crossed to obtain male YAP mice. flox / flox -Alb-cre + Mice, also known as YAP ΔHep Mice, in which the female-to-male ratio in the self-crossing of F1 generation mice was 1:3;

[0040] 1. Using PCR to target YAP flox / flox And Alb-Cre + Genotyping of mice

[0041] flox mouse primers:

[0042] F: 5'-GAACTCACATCTTTCCGGGTCCTG-3';

[0043] R: 5'-TTCGGACCAGTCTGACGGATCC-3';

[0044] The PCR amplification reaction system is shown in Table 1;

[0045] Table 1

[0046]

[0047] The PCR amplification reaction conditions are shown in Table 2.

[0048] Table 2

[0049]

[0050] Alb-cre mouse primers:

[0051] Alb-cre-F:5'-GAAGCAGAAGCTTAGGAAGATGG-3'; Alb-cre-R:5'-TTGGCCCCTTACCATAACTG-3';

[0052] The PCR amplification reaction system is shown in Table 3;

[0053] Table 3

[0054]

[0055] The PCR amplification reaction conditions are shown in Table 4.

[0056] Table 4

[0057]

[0058]

[0059] The results of the identification are as follows Figure 1 As shown

[0060] Depend on Figure 1 It can be seen that YAP flox / flox Homozygous: PCR product size is 351bp; Wild-type: PCR product size is 289bp. In addition, when Alb-cre-F and Alb-cre-R primers are used for PCR amplification, the product containing the Alb-cre gene is 390bp, while wild-type has no band.

[0061] 2. Detection of YAP using Western blotting and immunofluorescence staining flox / flox and YAP ΔHep Expression of YAP in mouse hepatocytes

[0062] Liver tissue was isolated, digested using protein lysis buffer, and total protein was extracted. After denaturation with loading buffer, proteins were separated by SDS-PAGE electrophoresis, transferred to PVDF, incubated overnight at 4°C with rabbit-derived YAP antibody, and then incubated for 1 hour at room temperature with HRP-labeled goat anti-rabbit IgG (H+L). YAP protein was detected using chemiluminescence immunoassay. The results are as follows: Figure 2 As shown;

[0063] Depend on Figure 2It can be seen that wild type and YAP flox / flox YAP protein is highly expressed in mouse liver, and YAP ΔHep No expression of YAP protein was observed in the middle jiao;

[0064] 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, and then incubated overnight at 4°C with goat-derived albumin (Albumin, Alb) antibody and rabbit-derived YAP antibody, respectively. They were then incubated at room temperature for 2 hours with Alexa Fluor 488-labeled donkey anti-goat secondary antibody and Cy3-labeled donkey anti-rabbit secondary antibody, followed by DAPI staining for 20 minutes. The sections were mounted with glycerol (an anti-quencher), and observed and imaged using a laser confocal microscope. The results are as follows: Figure 3 As shown;

[0065] Depend on Figure 3 It was found that all mouse liver Alb (hepatocyte marker protein) antibodies showed a green fluorescent signal, while YAP showed a red fluorescent signal. Wild-type and YAP... flox / flox Strong red fluorescence signal was observed in mouse hepatocytes, indicating that YAP was highly expressed in hepatocytes and was not knocked out. ΔHep No red fluorescent signal was observed in mouse hepatocytes, indicating that YAP was successfully knocked out in hepatocytes.

[0066] (b) YAP was fed a Lieber-DeCarli fluid diet. ΔHep The mice were fed for 5 days;

[0067] (c) YAP was fed a Lieber-DeCarli alcoholic fluid diet (5% alcohol). ΔHep Mice were fed for 10 days, and after the feeding period ended, they were given an alcohol gavage of 5 g / kg body weight once to obtain the mouse model of late-stage ALD-related liver fibrosis.

[0068] Example 2

[0069] This example validates the mouse model of late-stage ALD-related liver fibrosis from Example 1:

[0070] In step (c) of the construction method of Example 1, the animals were euthanized 8 hours after gavage and samples were taken for testing;

[0071] 1) Oil Red O staining of frozen liver sections

[0072] (1) Preparation of Oil Red "O"

[0073] a. Dissolve 0.5 g of Oil Red "O" dry powder in a small amount of isopropanol, then add isopropanol to make up to 100 mL to prepare a stock solution, and store at 4°C protected from light.

[0074] b. When using, dilute the stock solution with deionized water at a ratio of 3:2, filter with qualitative filter paper, prepare the working solution, and use it within 1 hour.

[0075] (2) Oil Red "O" staining of frozen liver sections

[0076] a. Take frozen sections of liver, thaw for 20 min, fix with 4% paraformaldehyde for 15 min, wash with deionized water 3 times, 5 min each time;

[0077] b. Immerse the sections in Oil Red "O" working solution for 30 min, then wash with deionized water 3 times, 5 min each time;

[0078] c. Counterstain with hematoxylin for 6 minutes, then gently rinse with tap water for 5 minutes;

[0079] d. Glycerin gelatin sealing sheet;

[0080] e. Image acquisition using a microscope.

[0081] The collection results are as follows Figure 4 As shown; by Figure 4 It can be seen that after 15 days of Gao-Binge alcohol diet feeding, YAP flox / flox Slight lipid accumulation was observed in the hepatocytes of mice, while YAP ΔHep Lipid accumulation and a significant increase in lipid droplets were observed in mouse hepatocytes, indicating marked fatty degeneration of the liver.

[0082] 2) Liver tissue was obtained, embedded in OCT, sectioned (thickness: 7μm), and fixed. Immunofluorescence staining with CD68 (macrophage marker protein) and MPO (neutrophil marker protein) was performed respectively.

[0083] 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 overnight at 4°C with rat-derived CD68 antibody and rabbit-derived MPO antibody, respectively, and incubated at room temperature for 2 hours with Cy3-labeled goat anti-rat or rabbit anti-antibody. The nuclei were stained with DAPI for 20 minutes, mounted with anti-quenching agent glycerol, and observed and imaged using a laser confocal microscope.

[0084] The results are as follows Figure 5 and 6 As 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 Less infiltration, while YAPΔHep Mouse liver macrophages ( Figure 5 ) and neutrophils ( Figure 6 Increased infiltration.

[0085] 3) Liver tissue was taken for OCT embedding, sectioning (thickness: 7μm), and fixation, and stained with Sirius red.

[0086] Frozen sections (7 μm) of liver tissue were taken and equilibrated at room temperature for 10-15 min. They were then fixed overnight in 10% formalin at room temperature, rinsed with running water, and dried. They were stained with Sirius red for 1 h, rinsed with running water for 6 min, stained with hematoxylin for 2 min, washed with running water for 6 min, and dehydrated in a gradient of 80%, 90%, 95%, and 100% ethanol. The sections were cleared with xylene, mounted with neutral resin, and images were acquired and analyzed under a microscope.

[0087] The results are as follows Figure 7 As shown; by Figure 7 It can be seen that after 15 days of Gao-Binge alcohol diet feeding, YAP flox / flox The mice had milder liver fibrosis, while YAP ΔHep The degree of liver fibrosis in mice was aggravated.

[0088] 4) Liver tissue was taken, embedded in OCT, sectioned (thickness: 7μm), and fixed, and then α-actin (hepatic stellate cell marker protein) immunofluorescence staining was performed.

[0089] 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 Cy3-labeled α-actin at room temperature for 2 hours, stained with DAPI for 20 minutes, mounted with glycerol as an antiquencher, and observed and imaged using a laser confocal microscope.

[0090] The results are as follows Figure 8 As 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, while YAP ΔHep The liver of mice contains a large number of hepatic stellate cells, mostly located in the central vein.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A method for constructing an ALD late-stage associated liver fibrosis mouse model, characterized in that, The construction method comprises the following steps: (a) Homozygous mice YAP flox / flox were crossed with Alb-Cre + mice to obtain F1 mice YAP flox / - -Alb-Cre + ; F1 mice were interbred to obtain male YAP flox / flox -Alb-cre + mice, denoted as YAP ΔHep mice; (b) YAP was fed according to the Gao-binge alcohol diet method ΔHep The mice were fed, and after the feeding was completed, alcohol was used for gavage, and the ALD late-stage related liver fibrosis mouse model was obtained.

2. The construction method of claim 1, wherein, In the step (a), the homozygous mouse YAP flox / flox The 3rd exon of the YAP gene is inserted with loxp sites at both ends.

3. The construction method of claim 1, wherein, In step (a), homozygous mouse YAP flox / flox Alb-Cre + The male to female ratio of the mice is 1: (1-5).

4. The construction method of claim 1, wherein, In the step (a), the ratio of male to female in the self-crossing of the F1 generation mice is 1: (1-5).

5. The construction method of claim 1, wherein, In the step (b), the Gao-binge alcohol diet method comprises: First, adaptive feeding is carried out by using the Gao-binge fluid feed, and then Gao-binge fluid feed containing 5% alcohol is used for continuous feeding for a period of time.

6. The construction method of claim 5, wherein, The adaptive feeding time is 3-8 days.

7. The construction method of claim 5, wherein, The continuous feeding time is not less than 10 days.

8. The construction method of claim 1, wherein, The YAP ΔHep Mice were 10-12 weeks of age.

9. The construction method of claim 1, wherein, In the step (b), the gavage frequency is 1 time, and the gavage amount of alcohol is 4-6 g / kg.

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