Construction method of metabolism-related fatty liver disease combined chronic hepatitis B-related hepatocellular carcinoma model

Abstract

The invention discloses a construction method of a metabolism-related fatty liver disease and chronic hepatitis B-related hepatocellular carcinoma model, and belongs to the technical field of disease animal models. The construction method comprises the following steps: S1, linearizing an HBV genome, and injecting the linearized HBV genome into a fertilized egg of a C57BL / 6 mouse to establish an HBV transgenic mouse; and S2, feeding the HBV transgenic mouse in the step S1 with D09100310 feed to obtain the mouse model of metabolism-related fatty liver disease combined with chronic hepatitis B-related hepatocellular carcinoma. According to the construction method of the metabolism-related fatty liver disease combined chronic hepatitis B related hepatocellular carcinoma model, HBV-Tg mice are selected as experimental animals, the D09100310 mouse feed is utilized to construct the metabolism-related fatty liver disease combined chronic hepatitis B related hepatocellular carcinoma model, the stability and repeatability are good, objectiveness and simplicity are achieved, operability is high, and the model is suitable for being used for the clinical application. And other possible interference factors are reduced.

Description

Technical Field

[0001] The present invention relates to the field of disease animal models, and in particular to a method for constructing a metabolism-related fatty liver disease combined with chronic hepatitis B-related hepatocellular carcinoma model. Background Art

[0002] Liver cancer is the sixth most common malignant tumor in the world and the third leading cause of death from malignant tumors. Hepatocellular carcinoma (HCC) is the most common type of liver cancer, accounting for about 80% of all liver cancer cases. The introduction of a new nomenclature for fatty liver disease, metabolic-associated fatty liver disease (MAFLD), has made metabolic-associated fatty liver disease no longer an exclusionary diagnosis, but based on the presence of metabolic dysfunction, so it can now coexist with other liver diseases. Chronic hepatitis B virus (HBV) infection affects approximately 296 million people worldwide and is the leading cause of cirrhosis and liver cancer worldwide.

[0003] HBV can promote the development of HCC through direct and indirect mechanisms, including direct destruction and indirect immune-induced damage. At the same time, the integration of HBV DNA genome with host genes, host gene instability, mutation of oncogenes and tumor suppressor genes, and expression of mutant proteins can lead to the occurrence of cancer. Related studies on MAFLD-HCC also suggest that mechanisms including abnormal activation of multiple signaling pathways can promote the transformation of MAFLD patients to HCC.

[0004] In Asia, especially in East Asia, there is a background of HBV infection, which is complicated by MAFLD in middle-aged and elderly people, and further progresses to HCC, which is relatively consistent with the epidemiological status of liver cancer in the region. However, there are currently no experimental animals that are relatively consistent with this pathogenesis and suitable for observing a large number of samples. The main reason for this problem is that non-primates have little or no susceptibility to HBV virus, but the emergence of HBV-Tg transgenic mice has made it possible to study HBV infection in non-primate experimental animals.

[0005] As a model organism, mice are widely used in constructing disease models due to their low breeding cost, strong regeneration ability, and relatively close relationship with humans as mammals. The present invention proposes a new method for constructing a metabolic-related fatty liver disease combined with chronic hepatitis B-related hepatocellular carcinoma model using special feed induction. Summary of the invention

[0006] The purpose of the present invention is to provide a method for constructing a model of metabolic-related fatty liver disease combined with chronic hepatitis B-related hepatocellular carcinoma. HBV-Tg mice are selected as experimental animals, and a high-fat, high-fructose, high-cholesterol feed is used to construct a model of metabolic-related fatty liver disease combined with chronic hepatitis B-related hepatocellular carcinoma. The construction method has good stability and repeatability, is objective and simple, has strong operability, and reduces other possible interference factors.

[0007] To achieve the above objectives, the present invention provides a method for constructing a metabolic-related fatty liver disease combined with chronic hepatitis B-related hepatocellular carcinoma model, comprising the following steps:

[0008] S1, linearize the HBV genome and inject it into C57BL / 6 mouse fertilized eggs to establish HBV transgenic mice C57BL / 6N-Tg 6 / Vst;

[0009] S2. Feed the HBV transgenic mice C57BL / 6N-Tg6 / Vst in step S1 with a high-fat, high-fructose, and high-cholesterol feed to obtain a mouse model of metabolic-related fatty liver disease combined with chronic hepatitis B-related hepatocellular carcinoma.

[0010] Preferably, HBV DNA and HBsAg can be detected in the serum and liver tissue of HBV transgenic mice.

[0011] Preferably, the HBV genome is GenBank No. AF305422.1.

[0012] Preferably, in step S2, the rearing conditions are: temperature 28.5°C, 12 hours light / 12 hours dark.

[0013] Preferably, in step S2, the high-fat, high-fructose, high-cholesterol feed is fed in a self-feeding manner.

[0014] Therefore, the method for constructing a metabolic-related fatty liver disease combined with chronic hepatitis B-related hepatocellular carcinoma model of the present invention has the following beneficial effects:

[0015] (1) The present invention adopts a non-invasive method to construct an animal model of metabolic-related fatty liver disease combined with chronic hepatitis B-related hepatocellular carcinoma, and applies a high-fat, high-fructose, and high-cholesterol feed to construct an animal model of metabolic-related fatty liver disease combined with chronic hepatitis B-related hepatocellular carcinoma. Under the condition of reducing the harm to the animal model, the animal model of metabolic-related fatty liver disease combined with chronic hepatitis B-related hepatocellular carcinoma is successfully established by the experimental animals taking the high-fat, high-fructose, and high-cholesterol feed orally on their own initiative;

[0016] (2) The construction method of the present invention has good stability and repeatability, is objective and simple, has strong operability, reduces other possible interference factors, and provides a necessary experimental basis for studying the relevant mechanisms and development of treatment methods for metabolic-related fatty liver disease combined with chronic hepatitis B-related hepatocellular carcinoma;

[0017] (3) The present invention uses HBV-Tg mice to construct a model of metabolic-related fatty liver disease combined with chronic hepatitis B-related hepatocellular carcinoma. Mice, as model animals, have the advantages of relatively low breeding costs, strong reproductive ability, and relatively close relationship with humans as mammals. Compared with primates, mice have absolute advantages in the ease of breeding and modeling. In addition, mice develop rapidly and can quickly simulate the pathological processes and treatments of related human diseases.

[0018] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0020] Figure 1 Flow chart of constructing an animal model of metabolic-related fatty liver disease combined with chronic hepatitis B-related hepatocellular carcinoma for the present invention;

[0021] Figure 2 Comparison of B-ultrasound images between the model group and the blank control group in the embodiment of the present invention;

[0022] Figure 3 The figure is a comparison of B-ultrasound images of mice in the model group and the blank control group at the end of the experiment. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0024] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0025] The instruments, equipment, reagents and materials used in the examples were all obtained through commercial channels.

[0026] Example

[0027] A method for constructing a metabolic-related fatty liver disease combined with chronic hepatitis B-related hepatocellular carcinoma model, the process is as follows Figure 1 As shown, the specific steps include:

[0028] S1. Preparation of HBV-Tg male mice: Male WT C57BL / 6 mice were purchased from Beijing Vita Biotechnology Co., Ltd. The linearized HBV genome (Gentype A, GenBank ID: AF305422.1, subtypeadw2) was injected into the fertilized eggs of C57BL / 6 mice by pronuclear microinjection to establish the HBV transgenic mouse model C57BL / 6N-Tg (1.28merHBV)6 / Vst (i.e., HBV-Tg mice). After a 12 / 12 hour light / dark cycle, the mice were placed in a controlled environment and the ambient light was adjusted.

[0029] S2. Experimental Grouping and Treatment

[0030] Model group: 10-month-old male mice were fed with D09100310 feed;

[0031] Blank control group: 10-month-old male mice were fed with normal mouse feed.

[0032] The rearing conditions were: temperature 28.5°C, 12 h light / 12 h dark.

[0033] The D09100310 mice were fed with feed on their own until they developed metabolic-related fatty liver disease and chronic hepatitis B-related hepatocellular carcinoma.

[0034] D09100310 mouse feed is a specially formulated mouse feed developed by Research Diets, Inc. It is a high-fat, high-sugar, high-cholesterol diet feed, and is mainly used to induce animal models of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). It can induce the NAFLD / NASH model in C57BL / 6 mice. When feeding C57BL / 6 mice, it induces fatty degeneration at 12-16 weeks, inflammasomes at 20-26 weeks, and fibrosis at 26-34 weeks, while simulating the entire pathological process of human NASH, including the characteristics of metabolic syndrome.

[0035] S3. After the experimental animals were fed with D09100310 mouse diet, liver steatosis and intrahepatic tumor formation were evaluated by mouse ultrasound every month.

[0036] After the mice were anesthetized with sevoflurane inhalation anesthesia, the upper abdomen of the mice was shaved, and the liver area was observed using a color Doppler ultrasound imager. After debugging, B-ultrasound images of the mouse liver area were obtained respectively. The nodule size was measured using a Doppler ultrasound imager, and the grayscale value was measured using Image J.

[0037] like Figure 2 The figure shows the comparison of B-ultrasound images of the model group and the blank control group. The comparison shows that the model group had a low-density liver shadow at 7 weeks, which gradually increased over time, considering hepatocellular carcinoma.

[0038] like Figure 3 The figure shows the comparison of B-ultrasound images of all mice in the model group and the blank control group at the end of the experiment. The blank control group showed B-ultrasound images at 30 weeks, while (1), (3), and (5) in the model group showed B-ultrasound images at 30 weeks, (2) showed images at 26 weeks, and (4) showed images at 17 weeks. This is because according to animal ethics requirements, mice (2) and (4) developed tumors earlier, and reached the humane endpoint of the animal experiment when their body weight decreased by more than 15%. By comparison, all mice in the model group had liver tumors, but no tumors were found in the blank control group.

[0039] The comparison of gray values ​​between the model group and the blank control group is shown in Table 1:

[0040] Table 1 Gray values ​​of model group and blank control group

[0041]

[0042]

[0043] It can be seen from the above table that the grayscale value of the liver is diffusely increased under B-ultrasound. Combined with the B-ultrasound image, it can be judged that the model of metabolic-related fatty liver disease combined with chronic hepatitis B-related hepatocellular carcinoma has been successfully constructed.

[0044] Therefore, the method for constructing a metabolic-related fatty liver disease combined with chronic hepatitis B-related hepatocellular carcinoma model of the present invention obtains a corresponding mouse model through simple feeding induction, and the overall condition of the obtained modeled mice is relatively good, which provides a good model animal for studying metabolic-related fatty liver disease combined with chronic hepatitis B-related hepatocellular carcinoma. At the same time, this model also provides a good tool for the study of pathogenesis and treatment plans.

[0045] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for constructing a model of metabolic-related fatty liver disease combined with chronic hepatitis B-related hepatocellular carcinoma, characterized in that: The steps include: S1, linearize the HBV genome and inject it into C57BL / 6 mouse fertilized eggs to establish HBV transgenic mice C57BL / 6N-Tg 6 / Vst; S2. Feed the HBV transgenic mice C57BL / 6N-Tg 6 / Vst in step S1 with a high-fat, high-fructose, and high-cholesterol feed to obtain a mouse model of metabolic-related fatty liver disease combined with chronic hepatitis B-related hepatocellular carcinoma.

2. The method for constructing a metabolic-related fatty liver disease combined with chronic hepatitis B-related hepatocellular carcinoma model according to claim 1, characterized in that: HBV DNA and HBsAg can be detected in the serum and liver tissues of HBV transgenic mice.

3. The method for constructing a metabolic-related fatty liver disease combined with chronic hepatitis B-related hepatocellular carcinoma model according to claim 1, characterized in that: The HBV genome is GenBank No. AF305422.

1.

4. The method for constructing a metabolic-related fatty liver disease combined with chronic hepatitis B-related hepatocellular carcinoma model according to claim 1, characterized in that: In step S2, the rearing conditions are: temperature 28.5°C, 12 hours light / 12 hours dark.

5. The method for constructing a metabolic-related fatty liver disease combined with chronic hepatitis B-related hepatocellular carcinoma model according to claim 1, characterized in that: In step S2, the high-fat, high-fructose, high-cholesterol feed is fed by autonomous feeding.

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