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Hepatitis virus in-vitro culture model as well as construction method and application thereof

A hepatitis virus, in vitro culture technology, applied in the direction of virus/phage, virus, retro DNA virus, etc., can solve the problems of low infectivity, high homogeneity, instability, etc., to achieve good repeatability, simple method, scalable applied effect

Inactive Publication Date: 2010-12-15
INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The research status of HCV in vitro cell culture model is as follows: 1. Liver-derived cell models include primary liver cells and fetal liver cells, but because of their low replication level and instability, they cannot meet the requirements of drug screening research; liver cancer cell lines cannot for the study of infection mechanisms
2HCV genome transfection model: including Huh-7 cells transfected with HCV JFH-1 RNA, fetal liver cells transfected with HCV RNA without adaptive mutation, but the defect is that only HCV 2a and 1a can be subcultured, and the virus secreted by cell culture High homogeneity and low infectivity of particles compared to animal experiments
[0010] So far, there has been no report on the use of human fetal liver stem cells to construct HBV and HCV in vitro culture models

Method used

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  • Hepatitis virus in-vitro culture model as well as construction method and application thereof
  • Hepatitis virus in-vitro culture model as well as construction method and application thereof
  • Hepatitis virus in-vitro culture model as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Embodiment 1 constructs hepatitis virus in vitro culture model such as Figure 1-Figure 4 shown

[0068] I. Isolation and culture of human fetal liver stem cells:

[0069] (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% DNaseI, 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 / ...

Embodiment 2

[0098] I.HCV high copy positive serum infects human fetal liver stem cells: HCV (10 5 -10 7 Copies) serum 100ul was inoculated into six-well plate cells in 1ml DMEM / F12 culture medium (serum-free); after incubation at 37°C for 24h, the supernatant was sucked out, and the cells were cultured in DMEM / F12 culture medium (10% FBS). HCV can be obtained by taking the supernatant within 48 hours, which can be sampled continuously for 5 times.

[0099] II. Verify that hepatitis C virus has infected human fetal liver stem cells and multiplied and continuously secreted outside the cells:

[0100] Detection of virus titers in cell supernatants at different times by fluorescent quantitative PCR

[0101] (1). RNA extraction: RNA was extracted using the Hepatitis C virus nucleic acid quantitative detection kit and stored at -20°C. The negative and positive quality control samples were treated in the same way for later use.

[0102] (2). Fluorescent quantitative PCR amplification: using t...

Embodiment 3

[0111] Example 3 as Figure 6 As shown, the difference with embodiment 1 is as follows:

[0112] 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, 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. Cel...

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Abstract

The invention relates to a hepatitis virus in-vitro culture model as well as a construction method and application thereof. The hepatitis virus in-vitro culture model is constructed by adopting human fetal liver stem cells and comprises the steps of carrying out separate culture on human fetal liver stem cell, immunohistochemically identifying human fetal liver stem cell and infecting human fetal liver stem cell with hepatitis virus by blood serum and verifying whether hepatitis virus infects the human fetal liver stem cells or not and the virus propagate and continuously secrete outside the cell or not; the model can be used for the screening and effect evaluation of an in-vitro hepatitis virus resistant medicament; the human fetal liver stem cells used in the hepatitis virus in-vitro culture model can be infected with hepatitis virus and can continuously secrete hepatitis virus; the human fetal liver stem cells can be subcultured; and the invention has simple and convenient method and high repeatability.

Description

technical field [0001] The invention relates to an in vitro culture model and its construction method and application, especially the hepatitis virus in vitro culture model and its construction method and application. 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 steps in studying the biological characteristics of HBV, the pathogenesis ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735C12Q1/70C12Q1/68C12Q1/02
CPCC12N2770/24251C12N7/00C12N2730/10151
Inventor 李君文金敏郭向飞王姝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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