Construction method of metabolism-related fatty liver cancer mouse model

Abstract

The invention discloses a construction method of a metabolism-related fatty liver cancer (MAFLD liver cancer) mouse model, and relates to the technical field of biology. The specific construction method comprises the following steps: feeding a 7-9-week-old mouse with a high-fat and high-sugar diet for 24 weeks, inducing the mouse to form fatty liver, and causing metabolic dysfunction; injecting carbon tetrachloride into the abdominal cavity of the mouse once a week while feeding the mouse with the high-fat and high-sugar diet, and inducing the liver of the mouse to form liver cancer continuously for 24 weeks, so as to construct the MAFLD liver cancer mouse. The mouse liver constructed by the method has metabolic dysfunction, fatty liver and liver cancer lesions, and has extremely high application value in research and development of drugs for researching MAFLD liver cancer pathogenesis and fatty liver related liver cancer in the future.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for constructing a metabolism-related fatty liver cancer mouse model. Background Art

[0002] Hepatitis B virus infection is the main cause of liver cancer. However, with the development of the global economy, the living standards of residents have been improving year by year, and the incidence of metabolic-related diseases (such as fatty liver, obesity, diabetes, hyperlipidemia, etc.) has shown a clear upward trend. Metabolic associated fatty liver disease (MAFLD) may eventually induce liver-related complications such as non-alcoholic steatohepatitis (NASH), cirrhosis and liver cancer. Recent epidemiological data show that the number of patients with MAFLD-related liver cancer (MAFLD liver cancer) is increasing year by year. Therefore, studying the pathogenesis and prevention and treatment strategies of MAFLD liver cancer is one of the current hot topics in liver disease research at home and abroad.

[0003] The pathogenesis of MAFLD liver cancer is still not fully understood, and in-depth research and verification are required through a large number of animal experiments. At present, there are relatively few methods for constructing MAFLD liver cancer animal models at home and abroad, and the standards are not unified, which greatly limits the in-depth research on the pathogenesis and therapeutic drugs of MAFLD-related liver cancer. The existing methods for constructing small animal models of MAFLD liver cancer include transgenic induction, cell implantation, and chemical damage induction. These methods have their own limitations. Specifically: (1) The construction cost of transgenic induced mouse models is high, and the model is unstable. Moreover, since the model is induced by transgenic, it cannot reproduce liver fat and hepatocellular carcinoma, which is far from the pathogenesis of human MAFLD liver cancer and lacks the model parameters required for MAFLD liver cancer research and new drug development. (2) The limitations of cell implantation are in vitro cell culture and the survival rate of cells after inoculation. At present, there are relatively few scientific research teams that have successfully cultured MAFLD liver cancer cells in vitro at home and abroad, and the technical information has not been made public, making it difficult to obtain. Related reports include Wong AM (Wong AM, Ding X, Wong AM, et al. Unique molecular characteristics of NAFLD-associated liver cancer accentuate β-catenin / TNFRSF19-mediated immune evasion. J Hepatol 2022; 77: 410-423) et al. cultured MAFLD liver cancer cells in vitro and implanted them in mice. (3) The mouse model induced by chemical damage is currently the most commonly used model. However, different chemical reagents and construction methods lead to great heterogeneity of mouse models. For example, Asgharpour A (Asgharpour A, Cazanave SC, Pacana T, et al. A diet-induced animal model of non-alcoholic fatty liver disease and hepatocellular cancer. J Hepatol 2016; 65: 579-88) and others induced a NASH liver cancer model by hybridizing C57BL / 6J and 129S1 / SvImJ mice and combining a high-fat and high-sugar diet, but the modeling cycle of this model is long, 52 weeks. In addition, the genetic information of hybrid mice is different from that of simple C57BL / 6J mice, and it is difficult to stably inherit them; while the genetic information of related factors and signaling pathways involved in the occurrence and development of MAFLD liver cancer is similar to that of C57BL / 6J. In addition, during the construction of the existing model, mice are prone to death during feeding, which is not suitable for the needs of simple and rapid modeling.

[0004] In summary, the purpose of the present invention is to provide a method for constructing a metabolism-related fatty liver cancer mouse model. Summary of the invention

[0005] In view of the above shortcomings, the present invention provides a method for constructing a metabolic-related fatty liver cancer mouse model, and obtains a mouse model that is most similar to the clinical indications of human MAFLD liver cancer. The model can be used for in vivo experiments on fatty liver, metabolic-related diseases and liver cancer-related mechanisms, and the development of new drugs. It has extremely high application value in the pathogenesis and drug research of MAFLD liver cancer. The specific technical scheme is as follows:

[0006] A method for constructing a metabolic-related fatty liver cancer mouse model comprises the following steps: feeding mice with a Western diet and high-fructose and high-glucose drinking water, combined with intraperitoneal injection of carbon tetrachloride once a week, until obtaining MAFLD liver cancer mice;

[0007] The Western diet contained 41% sucrose and 1.25% cholesterol;

[0008] In the high-fructose and high-glucose drinking water, the concentration of D-fructose is 15.0-25.0 g / L, and the concentration of D-glucose is 15.0-20.0 g / L;

[0009] The injection dosage of carbon tetrachloride is based on the weight of mice, and each injection dosage is 0.1-0.2 μl / g. Before each injection, it is dissolved in 30-50 μl of corn oil.

[0010] Furthermore, the mice are male C57BL / 6J mice aged 7 to 9 weeks.

[0011] Furthermore, the feeding period of the Western diet feed is 24 weeks, with free intake every day.

[0012] Furthermore, the high-fructose and high-glucose drinking water is fed for 24 weeks, and is freely available for drinking every day.

[0013] Furthermore, the injection time of the carbon tetrachloride is 24 weeks.

[0014] Furthermore, the construction method also includes the following steps: killing the mice after 24 weeks of feeding, and identifying MAFLD-related liver cancer pathology through blood biochemistry, liver function and liver pathology.

[0015] The present invention also provides a mouse model of metabolism-related fatty liver cancer, which is constructed using the above method, and the mouse model has pathological characteristics of MAFLD-related liver cancer.

[0016] The present invention also provides applications of the above-mentioned metabolism-related fatty liver cancer mouse model, specifically applications in screening drugs for preventing or treating fatty liver-related liver cancer, and applications in research on metabolism-related fatty liver cancer.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The present invention uses Western diet feed and high-fructose and high-glucose drinking water, combined with carbon tetrachloride, to successfully construct a MAFLD-related liver cancer mouse model, which is most similar to the clinical indications of human MAFLD liver cancer. Among them, 7-9 week-old mice were fed a high-fat and high-sugar diet for 24 weeks to induce fatty liver and metabolic dysfunction in mice; while feeding the mice a high-fat and high-sugar diet, the mice were intraperitoneally injected with carbon tetrachloride once a week for 24 consecutive weeks to induce liver cancer in the mice. This model can be widely used in basic and clinical research, which is conducive to monitoring the biological behavior of metabolic-related fatty liver disease developing into liver cancer; it is conducive to monitoring the sensitivity of immune drugs and biological targeted drug treatments for MAFLD-related liver cancer, and providing a reference for the best treatment plan; it is conducive to using the non-human animal model of MAFLD-related liver cancer for screening drugs to prevent or control liver cancer caused by metabolic-related fatty liver disease.

[0019] 2. Compared with the chemical injury-induced mouse model (the tumor formation rate is 70% at 52 weeks of modeling) which is currently widely used, the present invention has a shorter modeling time and a higher success rate. The tumor formation rate exceeds 50% at 24 weeks of modeling, and the tumor formation rate can be increased to 85.0% at 32 weeks of modeling, and the mortality rate is lower (the mortality rate is only 4.76%), which is suitable for the current simple and fast modeling needs.

[0020] 3. The present invention reduces the mortality rate in the modeling process by controlling the injection concentration and injection dosage of carbon tetrachloride, and improves the success rate of modeling by controlling the ratio of sucrose and cholesterol content in the feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for describing the embodiments are briefly introduced below.

[0022] Figure 1 This is the trend curve of body weight changes of mice in each experimental group in Example 1 for 24 weeks.

[0023] Figure 2 The blood glucose change curves of the mice in each experimental group in Example 1 after four glucose tolerance tests, wherein: Figure 2 A is the trend of blood sugar changes in the glucose tolerance test of mice in the 6th week. Figure 2 B is the trend of blood glucose changes in the glucose tolerance test of mice at week 12. Figure 2 C is the trend of blood glucose changes in the glucose tolerance test of mice at week 18. Figure 2 D is the trend of blood glucose changes in the glucose tolerance test of mice at week 24.

[0024] Figure 3 These are the mouse serum insulin and glucagon levels at the experimental endpoints of each experimental group in Example 1.

[0025] Figure 4 These are the common ultrasound images of mice at the experimental endpoints of each experimental group in Example 1.

[0026] Figure 5 These are ultrasound contrast-enhanced images of mice at the experimental endpoints of each experimental group in Example 1.

[0027] Figure 6 These are the serum ALT and AST levels of mice at the experimental endpoints of each experimental group in Example 1.

[0028] Figure 7 This is the mouse serum AFP level at the experimental endpoint of each experimental group in Example 1.

[0029] Figure 8 This is the gross appearance and pathological HE staining of mouse liver at the experimental endpoint of each experimental group in Example 1. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present invention are described clearly and completely below. The described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] The following materials can be obtained from commercial sources, among which ordinary feed was purchased from Beijing Keao Xieli Feed Co., Ltd., and Western diet feed was purchased from Dyets Company.

[0032] Example 1

[0033] Construction of a mouse model of metabolic-related fatty liver cancer:

[0034] (1) 7-week-old male C57BL / 6J mice of uniform body weight were selected (purchased from Guangzhou Yancheng Biotechnology Co., Ltd.) and raised in an SPF environment. The mice were not treated in any way after purchase and were raised normally to adapt to the environment for 2 weeks.

[0035] (2) After the acclimatization, the animals were divided into the following groups according to the experimental plan:

[0036] ND+Oil group (experimental control group): 9-week-old male mice (n=10), fed with ordinary feed, drinking tap water, and intraperitoneally injected with 50ul corn oil once a week for 24 weeks.

[0037] ND+CCl4 group: 9-week-old male mice (n=10), fed with ordinary feed, drinking tap water, intraperitoneally injected with carbon tetrachloride (0.2μl / g) dissolved in 50ul corn oil once a week for 24 weeks.

[0038] WD+Oil group (simple fatty liver control group): 9-week-old male mice (n=10), fed with Western diet (containing 41% sucrose and 1.25% cholesterol), drinking high-glucose and high-fructose drinking water (the concentration of D-fructose was 23.1 g / L, the concentration of D-glucose was 18.9 g / L), and intraperitoneally injected with 50 ul corn oil once a week for 24 weeks of intervention.

[0039] WD+CCl4 group (experimental modeling group): 9-week-old male mice (n=42), fed with Western diet (containing 41% sucrose and 1.25% cholesterol), drinking high-glucose and high-fructose drinking water (the concentration of D-fructose was 23.1 g / L, and the concentration of D-glucose was 18.9 g / L), and intraperitoneally injected with carbon tetrachloride (0.2 μl / g) dissolved in 50 ul corn oil once a week for 24 weeks of intervention.

[0040] (3) At the end of the experiment, mice were anesthetized and underwent conventional ultrasound examination to preliminarily screen whether the mice had fatty liver and tumors in the liver. Some mice that met the screening criteria were anesthetized and underwent ultrasound angiography of the liver. Some mice that met the screening criteria were euthanized and their blood biochemistry, liver function, and liver pathology were tested to identify MAFLD-related liver cancer pathology.

[0041] The experimental results are as follows:

[0042] 1. The weight changes of mice in each experimental group during 24 weeks of feeding are as follows Figure 1 The results showed that the body weight of WD+CCl4 mice was higher than that of ND+Oil mice from week 12 to week 20, indicating that the mice had increased body weight.

[0043] 2. Changes in blood glucose in mice of each experimental group during the four glucose tolerance tests Figure 2 As shown, the results showed that WD+CCl4 mice showed obvious abnormal changes in glucose tolerance test compared with the control group mice, suggesting that WD+CCl4 mice had blood glucose metabolism dysfunction.

[0044] 3. The serum insulin and glucagon levels of mice in each experimental group at the end of the experiment Figure 3 As shown, the results showed that the serum insulin level of WD+CCl4 mice was significantly higher than that of the control group mice, suggesting that the mice had blood glucose metabolism disorder.

[0045] 4. The normal ultrasound images of mice at the end of each experimental group are as follows Figure 4As shown, the results showed that the liver area of ​​WD+CCl4 mice had high echo, which was consistent with the imaging diagnosis of fatty liver; tumors were detected in the liver of 20 mice (50.0%), indicating that WD+CCl4 mice had fatty liver and tumor formation.

[0046] 5. After screening the mice that meet the conditions in each experimental group, the liver ultrasound contrast images of the normal control group mice, the mice with simple fatty liver and the mice with fatty liver and tumors are as follows Figure 5 As shown, the results showed that there were significant differences in the entry and exit time curves of the contrast agent in the liver tumor area of ​​mice in the WD+CCl4 group, which was manifested as a faster entry time of the contrast agent in the liver tumor area, an earlier peak time, and an earlier start of the contrast agent withdrawal time, which was consistent with the sonographic manifestations of liver cancer, suggesting that the tumor was likely to be malignant.

[0047] 6. Example The serum ALT and AST levels of mice in each experimental group at the end of the experiment were as follows Figure 6 As shown, the results showed that the ALT level of mice in the WD+CCl4 group was significantly increased compared with that in the normal control group; the AST level of mice in the WD+CCl4 group was not significantly increased compared with that in the normal control group, indicating that the liver damage of the model animals was severe.

[0048] 7. Example The serum AFP level of mice at the end point of each experimental group was as follows Figure 7 As shown, the results showed that there was no significant difference in serum alpha-fetoprotein levels between the WD+CCl4 group mice and the control group mice, indicating that the model animals had no specificity in serum AFP indicators.

[0049] 8. Example The gross appearance and pathological HE staining of the mouse liver at the end point of each experimental group are as follows Figure 8 As shown, the results showed the formation of fatty liver and tumors in the liver of mice in the WD+CCl4 group; the pathological HE staining sections supported the diagnosis that the tumor was a malignant tumor.

[0050] A total of 42 C57BL / 6J mice were used in the experimental modeling group to construct a MAFLD-related liver cancer model, of which 2 mice died during the modeling process (mortality rate was 4.76%). 39 mice were diagnosed with fatty liver (fatty liver formation rate 97.5%) by ultrasound examination and pathological examination, and there were disorders in blood glucose metabolism and liver function, which were consistent with the diagnosis of metabolic-related fatty liver disease. Ultrasound examination of 20 mice revealed tumors in the liver (tumor formation rate was 50.0%). Further ultrasound angiography and pathological examination of the mouse liver tumors confirmed that the mouse liver tumors were consistent with the diagnosis of malignant tumors (liver cancer formation rate was 50.0%). We used the above modeling method to extend the experimental period to 32 weeks for another batch of 20 mice and found that the liver tumor rate increased to 85.0%.

[0051] In summary, the present invention uses Western diet feed and high fructose and high glucose drinking water, combined with carbon tetrachloride, to successfully construct a MAFLD-related liver cancer mouse model, which is most similar to the clinical indications of human MAFLD liver cancer. Among them, 7-9 week-old mice were fed a high-fat and high-sugar diet for 24 weeks to induce fatty liver in mice and metabolic dysfunction; while feeding the mice a high-fat and high-sugar diet, the mice were intraperitoneally injected with carbon tetrachloride once a week for 24 consecutive weeks to induce liver cancer in the mice. This model can be widely used in basic and clinical research, which is conducive to monitoring the biological behavior of metabolic-related fatty liver disease developing into liver cancer; it is conducive to monitoring the sensitivity of immune drugs and biological targeted drug treatments for MAFLD-related liver cancer, and providing a reference for the best treatment plan; it is conducive to the use of the non-human animal model of MAFLD-related liver cancer for screening drugs to prevent or prevent metabolic-related fatty liver disease leading to liver cancer. At the same time, compared with the currently used chemical damage-induced mouse model, the present invention has a shorter modeling time and a higher success rate.

[0052] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A method for constructing a metabolic-related fatty liver cancer mouse model, characterized in that: The method comprises the following steps: feeding mice with a Western diet and high-fructose and high-glucose drinking water, and intraperitoneally injecting carbon tetrachloride once a week until obtaining MAFLD liver cancer mice; The Western diet feed contains 35-45% sucrose and 0.8%-1.5% cholesterol; In the high-fructose and high-glucose drinking water, the concentration of D-fructose is 15.0-25.0 g / L, and the concentration of D-glucose is 15.0-20.0 g / L; The injection dosage of carbon tetrachloride is based on the weight of mice, and each injection dosage is 0.1-0.2 μl / g. Before each injection, it is dissolved in 30-50 μl of corn oil.

2. The method for constructing a metabolic-related fatty liver cancer mouse model according to claim 1, characterized in that: The mice are male C57BL / 6J mice aged 7 to 9 weeks.

3. The method for constructing a metabolic-related fatty liver cancer mouse model according to claim 1, characterized in that: The Western diet was fed for 24 weeks with free access every day.

4. The method for constructing a metabolic-related fatty liver cancer mouse model according to claim 1, characterized in that: The high-fructose and high-glucose drinking water was fed for 24 weeks and the mice were free to drink it every day.

5. The method for constructing a metabolic-related fatty liver cancer mouse model according to claim 1, characterized in that: The injection time of the carbon tetrachloride is 24 weeks.

6. The method for constructing a metabolic-related fatty liver cancer mouse model according to claim 1, characterized in that: The construction method also includes the following steps: killing the mice after 24 weeks of feeding, and identifying MAFLD-related liver cancer pathology through blood biochemistry, liver function and liver pathology.

7. A mouse model of metabolic-related fatty liver cancer, characterized in that: The mouse model is constructed using the method according to any one of claims 1 to 6, and has pathological characteristics of MAFLD-related liver cancer.

8. Use of the mouse model of metabolism-related fatty liver cancer as claimed in claim 7 in screening drugs for preventing or treating fatty liver-related liver cancer, and in research on metabolism-related fatty liver cancer.

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