阅读说明：本技术 一种用四氯化碳诱导慢加急性肝衰竭小鼠模型的方法 (Method for inducing chronic acute hepatic failure mouse model by carbon tetrachloride ) 是由 林炳亮 熊静 于 2018-07-03 设计创作，主要内容包括：本发明涉及一种慢加急性肝衰竭小鼠模型诱导方法,尤其涉及一种用四氯化碳诱导慢加急性肝衰竭小鼠模型的方法,方法的步骤如下:分组方法,Balb/c近交系小鼠随机平均分为模型组和对照组；给药方法,将四氯化碳用橄榄油稀释至10%和50%的浓度,模型组小鼠按照5至8ml每kg体重持续腹腔注射10%四氯化碳,每周给药两次,连续给药8周；8周后按照5至8ml每kg体重给予一次腹腔注射50%四氯化碳；检测方法,对实验小鼠进行生化指标检测和肝脏病理学检查。有益效果：采用Balb/c近交系小鼠作为模型动物,其多可采用近交系,重复性更好,且相对于大鼠成本更低,操作更为简便。本模型更符合临床乙型肝炎病毒(HBV)相关慢加急性肝衰竭的疾病发展过程。(The invention relates to a slow acute hepatic failure mouse model induction method, in particular to a method for inducing a slow acute hepatic failure mouse model by carbon tetrachloride, which comprises the following steps of grouping, randomly and evenly dividing Balb/c inbred line mice into a model group and a control group; the administration method comprises diluting carbon tetrachloride with olive oil to 10% and 50% concentration, and performing intraperitoneal injection of 10% carbon tetrachloride to mice of model group according to 5-8 ml per kg body weight, twice per week for 8 weeks; after 8 weeks, 50% carbon tetrachloride is given to the abdominal cavity once per kg body weight according to 5 to 8 ml; the detection method comprises the step of carrying out biochemical index detection and liver pathological examination on the experimental mouse. Has the advantages that: the Balb/c inbred line mouse is used as a model animal, and the inbred line can be mostly adopted, so that the repeatability is better, the cost is lower compared with that of a rat, and the operation is simpler and more convenient. The model is more suitable for the clinical disease development process of Hepatitis B Virus (HBV) related chronic plus acute liver failure.)

1. A method for inducing a mouse model of chronic plus acute liver failure by carbon tetrachloride, which is characterized by comprising the following steps:

a. the grouping method comprises the following steps: randomly and evenly dividing Balb/c inbred line mice into a model group and a control group;

b. the administration method comprises the following steps: diluting carbon tetrachloride with olive oil to 10% and 50% concentration, and performing intraperitoneal injection of 10% carbon tetrachloride to 5-8 ml per kg body weight of model mice twice a week for 8 weeks; after 8 weeks, 50% carbon tetrachloride is given to the abdominal cavity once per kg body weight according to 5 to 8 ml; the control mice were injected intraperitoneally with the same dose of olive oil at the same time;

c. the detection method comprises the following steps: the experimental mice were subjected to biochemical index detection and liver pathology examination.

2. The method for inducing a mouse model of chronic plus acute liver failure by carbon tetrachloride according to claim 1, wherein the biochemical index detection method comprises: after the mice are anesthetized by ether, eyeballs are picked at different time points to take blood, the collected whole blood of the mice is centrifuged, and then supernatant is left, and the levels of alanine aminotransferase and aspartate aminotransferase are detected by adopting a biochemical detection kit.

3. The method of inducing a mouse model of chronic plus acute liver failure with carbon tetrachloride according to claim 2, wherein: the time points for blood taking of the eyeball are 0h, 6h, 12h, 24h and 48 h.

4. The method of inducing a mouse model of chronic plus acute liver failure with carbon tetrachloride according to claim 1, wherein the liver pathology examination method is: collecting liver tissue specimens at time-sharing points, fixing with 10% neutral formaldehyde, making paraffin sections, and staining with hematoxylin-eosin and sirius red.

5. The method of inducing a mouse model of chronic plus acute liver failure with carbon tetrachloride of claim 4, wherein: the time points for collecting the liver tissue specimens are 6h, 12h, 24h and 48 h.

6. The method of inducing a mouse model of chronic plus acute liver failure with carbon tetrachloride of claim 1, wherein: the mice in the model group were subjected to intraperitoneal injection of 10% carbon tetrachloride at a rate of 5ml per kg body weight twice a week for 8 weeks.

7. The method of inducing a mouse model of chronic plus acute liver failure with carbon tetrachloride of claim 1, wherein: after 8 weeks of administration, the model group mice were administered with 50% carbon tetrachloride by intraperitoneal injection once per kg body weight at 8 ml.

Technical Field

The invention relates to a method for inducing a chronic acute hepatic failure mouse model, in particular to a method for inducing the chronic acute hepatic failure mouse model by carbon tetrachloride.

Background

Animal liver damage is the result of various liver diseases, and the prevention and treatment of liver damage is still a serious topic at present. The method has important practical significance by establishing an experimental liver injury animal model, researching the occurrence mechanism of liver diseases, screening liver-protecting drugs and exploring the liver-protecting action principle.

Carbon tetrachloride (CCl)₄) Is widely used for inducing liver injury animal model, CCl₄After entering into organism, the free radical generated by metabolism is activated by cytochrome P450 in liver to generate trichloromethyl free radical and trichloromethyl peroxy radical, and attacks phospholipid molecule on liver cell membrane, so that cell membrane and endoplasmic reticulum membrane are subjected to chloralkalation and lipid peroxidation to damage cell membrane and organelle; can be covalently combined with membrane lipid and protein macromolecules to influence protein metabolism, destroy the integrity of membrane structure and function, increase calcium ion influx, influence normal physiological functions of cells, and finally cause soluble enzyme in hepatocyte cytoplasm to exude and cell death.

According to the reports of relevant documents, the Liuxu Hua and the like establish a slow acute hepatic failure rat model by using carbon tetrachloride, D-galactosamine and lipopolysaccharide, but the establishment cost of the rat model is high, and the operation difficulty is higher than that of a mouse.

Zhang Hui Yun and so on use carbon tetrachloride to combine D-galactosamine, lipopolysaccharide to induce chronic acute hepatic failure mouse model. The model has the defects that too many liver injury drugs are needed for modeling, more interference factors are caused for the subsequent research of the disease, and the independent factor of HBV virus is mostly used for inducing the onset of the disease in combination with the more common clinical HBV-related chronic plus acute liver failure.

Disclosure of Invention

In summary, the present invention needs to provide a method for inducing a chronic acute hepatic failure mouse model with carbon tetrachloride, and a more stable and reliable chronic acute hepatic failure mouse model can be obtained by optimizing the inducing method through optimizing experimental animals.

A method for inducing a chronic plus acute liver failure mouse model by carbon tetrachloride specifically comprises the following steps:

a. the grouping method comprises the following steps: randomly and evenly dividing Balb/c inbred line mice into a model group and a control group;

b. the administration method comprises the following steps: diluting carbon tetrachloride with olive oil to 10% and 50% concentration, and performing intraperitoneal injection of 10% carbon tetrachloride to 5-8 ml per kg body weight of model mice twice a week for 8 weeks; after 8 weeks, 50% carbon tetrachloride is given to the abdominal cavity once per kg body weight according to 5 to 8 ml; the control mice were injected intraperitoneally with the same dose of olive oil at the same time;

c. the detection method comprises the following steps: the experimental mice were subjected to biochemical index detection and liver pathology examination.

Preferably, the biochemical index detection method comprises the following steps: after the mice are anesthetized by ether, eyeballs are picked at different time points to take blood, the collected whole blood of the mice is centrifuged, and then supernatant is left, and the levels of alanine aminotransferase and aspartate aminotransferase are detected by adopting a biochemical detection kit.

Preferably, the blood taking time for the eyeball is 0h, 6h, 12h, 24h and 48 h.

Preferably, the pathological examination method of the liver comprises the following steps: collecting liver tissue specimens at time-sharing points, fixing with 10% neutral formaldehyde, making paraffin sections, and staining with hematoxylin-eosin and sirius red.

Preferably, the time points for collecting the liver tissue samples are 6h, 12h, 24h and 48 h.

Preferably, the model group mice were administered twice weekly for 8 weeks with continuous intraperitoneal injection of 10% carbon tetrachloride at 5ml per kg body weight.

Preferably, the model group mice are administered with 50% carbon tetrachloride intraperitoneally at 8ml per kg body weight 8 weeks after administration.

The invention has the beneficial effects that:

CCl is selected and used in the technical scheme₄As a liver injury medicament, the medicament has been widely used for inducing liver injury animal models.

In the aspect of animal selection, the technical scheme adopts Balb/c inbred line mice as model animals, so that experimental errors caused by individual differences can be reduced, and the mice can adopt inbred lines and have better repeatability. Meanwhile, the Balb/c inbred mouse has lower cost and simpler and more convenient operation compared with a rat.

The model is found to meet the survival rate, biochemistry and pathological changes of clinical chronic acute hepatic failure diseases by carrying out biochemical index detection and liver pathological examination on experimental mice. The establishment of the model provides feasible basis for pathogenesis of chronic and acute liver failure and search of an effective treatment method.

Compared with the related methods of establishing a chronic acute hepatic failure rat model by applying carbon tetrachloride, D-galactosamine and lipopolysaccharide and the like in Liuxuhua and inducing the chronic acute hepatic failure rat model by applying carbon tetrachloride, D-galactosamine and lipopolysaccharide to Huiyu, the animal grouping and administration method of the technical scheme is simpler, fewer in groups, shorter in experimental period and easier to operate.

Drawings

FIG. 1 shows the pathological change microscopic view of liver tissue.

A is the lower view of the hematoxylin-eosin staining mirror of the mouse liver at different time points of the control group and the model group (X200);

b is the observational view of the sirius red staining lens of the liver of the control group and the model group (X100).

FIG. 2 is a comparison of serum alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) levels in control and model mice.

In the figure, the dotted line is the survival curve of the control group mouse, and the solid line is the survival curve of the model group mouse.

Detailed Description

The following describes a method for inducing a chronic plus acute liver failure mouse model by carbon tetrachloride according to the present invention with reference to the accompanying drawings 1 to 2 and the specific embodiment.