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Construction method and use of model of hepatitis B virus infection in vivo

A hepatitis B virus, in vitro infection technology, applied in the direction of virus/bacteriophage, microbe-based methods, viruses, etc., can solve the problem that human fetal liver stem cells have not been seen to construct HBV in vitro infection model, etc., and the method is simple and reproducible , to expand the effect of the application

Inactive Publication Date: 2010-04-21
INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] So far, there has been no report on the use of human fetal liver stem cells to construct an HBV infection model in vitro

Method used

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  • Construction method and use of model of hepatitis B virus infection in vivo
  • Construction method and use of model of hepatitis B virus infection in vivo
  • Construction method and use of model of hepatitis B virus infection in vivo

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 as Figure 1-Figure 4 shown

[0051] I. Isolation and culture of human fetal liver stem cells:

[0052] (1). Isolation and culture of fetal liver stem cells: Perfuse the liver with collagenase in situ, stop the perfusion when the liver turns grayish white, carefully peel off the liver cell suspension with a small spatula, filter through a 100-mesh screen, blow with a pipette to fully disperse the cells . The cell suspension was centrifuged in D-Hank's solution at 2000r / min, 4°C for 5min. Discard the supernatant, suspend the precipitated cells with 2ml DMEM medium, add 50ml DMEM medium containing 0.1% Pronase E and 0.005% DNase I, and incubate at 37°C, 5% CO2 for 30min. Then the cells were transferred to a centrifuge tube and placed on ice for 20 min to precipitate the cells by using the hepatic stem cell adhesion. The cell suspension was removed, and the pelleted cells were centrifuged at 4°C, 2000r / min for 10min. Cells were suspended in PBS. Use PBS sol...

Embodiment 2

[0077] The difference between embodiment 2 and embodiment 1 is as follows:

[0078] Such as Figure 6 Shown:

[0079]I. Isolation and culture of human fetal liver stem cells: Isolation and culture of fetal liver stem cells: perfuse the liver with collagenase in situ, stop the perfusion when the liver turns grayish white, carefully peel off the liver cell suspension with a small spatula, filter it through a 100-mesh sieve, and blow it with a pipette Disperse the cells thoroughly. The cell suspension was centrifuged in D-Hank's solution at 2000r / min, 4°C for 5min. Discard the supernatant, suspend the precipitated cells with 2ml DMEM medium, add 50ml DMEM medium containing 0.1% Pronase E and 0.005% DNase I, 37°C, 5% CO 2 Incubate for 30min. Then the cells were transferred to a centrifuge tube and placed on ice for 20 min to precipitate the cells by using the hepatic stem cell adhesion. The cell suspension was removed, and the pelleted cells were centrifuged at 4°C, 2000r / min...

Embodiment 3

[0081] Embodiment 3 is tested with interferon IFN medicine as the criterion, as Figure 5 Shown:

[0082] The application of hepatitis B virus in vitro infection model, the described use of human fetal liver stem cells for the screening and effect evaluation of anti-hepatitis B virus drugs in vitro is to inoculate stable growth of human fetal liver stem cells in a six-well plate for 24 hours, and then It was incubated with the virus serum for 24 hours, the virus liquid was sucked off, washed 6 times with PBS, and the washed liquid was left for inspection; 200IU / ml, 500IU / ml, 1000IU / ml, 2000IU / ml of interferon IFN (chemical medicine or Chinese herbal medicine or bioengineering drug), in addition, the group without anti-hepatitis B virus drug was used as the positive control, and the group without virus and anti-hepatitis B virus drug was used as the negative control, and each group was repeated for three wells, and incubated at 37°C After 3 days, the supernatant was taken, and...

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Abstract

The invention discloses a construction method and use of a model of hepatitis B virus infection in vivo. The construction method of the model of hepatitis B virus infection in vivo, which uses stem cells of the human fetal liver in anti-hepatitis B virus medicament screening, comprises the following steps: separation and cultivation of the stem cells of the human fetal liver; immunohistochemical detection of the stem cells of the human fetal liver; hepatitis B virus serum infection of the stem cells of the human fetal liver; and verification of the infection of the stem cells of the human fetal liver by the hepatitis B viruses and the propagation of the viruses. The construction method, which uses the stem cells of the human fetal liver in the model of the infection in vivo, is used to screen and evaluate anti-hepatitis B virus medicaments. The method has the advantages that: the stem cells of the human fetal liver can be infected by the anti-hepatitis B viruses and constantly secrete anti-hepatitis B viruses; the stem cells of the human fetal liver can be passed and cultured; the method is simple and convenient; and the repeatability is desirable.

Description

technical field [0001] The invention relates to the construction of a hepatitis B virus infection model, in particular to a construction method and application of a hepatitis B virus in vitro infection model for human fetal liver stem cells used for screening anti-HBV drugs. Background technique [0002] As we all know, hepatitis B virus (HBV) is one of the most important causes of liver disease. About 350 million people in the world are chronic HBV carriers, and severe cases can lead to liver cirrhosis and primary liver cancer. Viral hepatitis B is widely prevalent and highly contagious. It is one of the most common infectious diseases in the world. Once a person is infected by hepatitis B virus, inflammatory lesions will occur in the liver, liver cells will be damaged, and it will cause great harm to the human body. Therefore, it is of great significance to study the pathogenesis of HBV and its prevention and control methods. [0003] One of the important links in the stu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735C12Q1/70C12R1/93
CPCC12N2770/24251C12N7/00C12N2730/10151
Inventor 金敏郭向飞李君文陈照立邱志刚谌志强王新为王景峰
Owner INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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