Construction method and application of age-dependent hepatitis B mouse model system of a kind of balb/c background

A technology of mouse model and construction method, which is applied in biochemical equipment and methods, pharmaceutical formula, recombinant DNA technology, etc., can solve the problems such as no hepatitis B mouse model system, and achieve moderate price, simple preparation, and easy operation The effect of simple method

Inactive Publication Date: 2019-06-07
中国人民解放军第四五八医院
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

[0006] However, to date, there is no age-dependent mouse model system for hepatitis B in the BALB / c background

Method used

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  • Construction method and application of age-dependent hepatitis B mouse model system of a kind of balb/c background
  • Construction method and application of age-dependent hepatitis B mouse model system of a kind of balb/c background
  • Construction method and application of age-dependent hepatitis B mouse model system of a kind of balb/c background

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Experimental program
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Embodiment 1

[0038] Rag-1 - / - / HBV mouse breeding: Rag-1 - / - Mice were crossed with HBV transgenic mice to obtain F1 generation Rag-1 + / - / HBV mice. The F1 generation of male and female individuals mated with each other to obtain the F2 generation, whose Rag-1 genotype may be Rag-1 + / + , Rag-1 + / - and Rag-1 - / - , from which the Rag-1 required for the experiment was screened out - / - / HBV mice are tested by flow cytometry to confirm whether they contain mature T / B lymphocytes, and the obtained mice are used for production and experimental research.

[0039] Rag-1 - / - / Identification of HBV mice: including Rag-1 gene detection and HBsAg detection.

[0040] Detection of Rag-1 gene: Rag-1 gene was detected by PCR method, 2-5 mm of mouse tail tissue was clipped, DNA was extracted with TissueDNA Kit kit, and PCR was performed with 3 primers using this as a template. The sequences of the three primers are as follows:

[0041] P1: 5'-GAGGTTCCGCTACGACTCTG-3';

[0042] P2: 5'-CCGGACAAGTTTT...

Embodiment 2

[0055]Preparation of mouse model: Separation of spleen lymphocytes from 8-10-week-old wild-type BALB / c mice was ground on a 200-mesh nylon cloth with a 10ml syringe plunger, the cell suspension was centrifuged at 1500rpm for 8min, the supernatant was discarded, and resuspended in 1×PBS Cells, carefully added to 40% and 70% percoll separation medium, centrifuged at 1500rpm for 20min, the centrifuge tube was divided into 6 layers, from top to bottom were PBS, dead cells, 40% percoll, living splenocytes, 70% percoll and red blood cells , aspirated live splenocytes, washed once with 1×PBS, counted, and adjusted the concentration. Infuse 1×10 via the tail vein 7 Lymphocytes were given to 20 days old, 5×10 7 Rag-1 - / - / HBV mice.

[0056] Pathological changes of the liver after cell reinfusion: 3 days after spleen cell reinfusion, the rats were killed to take the liver, fixed with 4% formaldehyde, conventional HE staining, adult Rag-1 - / - / HBV mouse liver has a large number of in...

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Abstract

The invention discloses a construction method of a BALB / c-background age-dependent hepatitis B mouse model system and an application thereof. In the model, BALB / c-background replicating form HBV transgenic mice are hybridized with Rag-1 gene knockout mice in the same background, thereby screening adult and young Rag-1<- / -> mice expressing HBsAg. Mouse spleen lymphocytes from wild non-sensitized mice in the same background are transfused back to the adult and young mice, thereby constructing age-dependent acute hepatitis B and chronic hepatitis B mouse models. The model can be used for exploring the cellular and molecular mechanism of virus removal and persistent virus infection and searching a novel target point for immunotherapy of the hepatitis B, thereby screening an anti-hepatitis B medicine. The construction method of the model has good repeatability and significant stability and reliability of detection indices of virology and biochemistry and pathology. The method is reasonable in cost and has good application prospect.

Description

technical field [0001] The invention relates to the technical field of animal model construction, in particular to a construction method and application of a BALB / c background age-dependent hepatitis B mouse model system. Background technique [0002] Hepatitis B virus (HBV) infection is still a relatively serious public health problem in my country. 95% of adults infected with HBV can be cleared naturally, while 90% of perinatal infection will become chronic, resulting in liver fibrosis and cirrhosis , and even liver cancer. At present, it is believed that the pathogenesis of hepatitis B is mainly the pathological changes of cellular immunity caused by the elimination of hepatitis B virus in the body, and the immune response of the body to the virus depends on the interaction of a series of immune active cells. The natural hosts of human hepatitis B virus are limited to humans, chimpanzees and tree shrews. Therefore, although chimpanzees are considered to be the most ideal ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85A01K67/027A61K35/76A61K49/00
CPCA01K67/0275A01K2207/12A61K35/76A61K49/0008C12N15/8509
Inventor 易学瑞袁有成孔祥平刘光泽
Owner 中国人民解放军第四五八医院
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