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Method for establishing HCV (hepatitis C virus) cell model by using tree shrew bone marrow mesenchymal stem cells

A technology of cell model and bone marrow mesenchyme, which is applied in the field of biomedicine and can solve the problems of liver cells that cannot be passaged

Active Publication Date: 2019-04-26
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to solve the deficiencies in the prior art, and to provide a method for establishing a HCV cell model using tree shrew bone marrow mesenchymal stem cells. It is easy to obtain and amplify, and still has multi-directional differentiation potential after continuous subculture and cryopreservation; it can be combined with various viral vectors for transfection of various genes, which can not only maintain the stability of its own genes, but also efficiently express all genes. The characteristics of the transfected gene will not only promote the differentiation of stem cells into hepatocytes, but also promote the intracellular infection of miR-122 and the two most critical receptors CD81 and Occludin (OCLN) of HCV into the bone marrow mesenchyme Mesenchymal stem cells, which solve the problems that primary hepatocytes cannot be passaged and the research on HCV of liver cancer cell lines is insufficient

Method used

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  • Method for establishing HCV (hepatitis C virus) cell model by using tree shrew bone marrow mesenchymal stem cells
  • Method for establishing HCV (hepatitis C virus) cell model by using tree shrew bone marrow mesenchymal stem cells
  • Method for establishing HCV (hepatitis C virus) cell model by using tree shrew bone marrow mesenchymal stem cells

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Experimental program
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Effect test

Embodiment 1

[0042] The method for establishing the HCV cell model by utilizing tree shrew bone marrow mesenchymal stem cells comprises the following steps:

[0043] First, the tree shrew bone marrow mesenchymal stem cells were infected with the OCLN lentiviral vector, then the CD81 lentiviral vector was added to continue the infection, and then the miR-122 lentiviral vector was added for infection. After 4 hours of infection, the volume of the current culture was added. Base volume 1 / 2 of fresh medium containing 2 μg / mL polybrene, the next day, remove the old medium, replace with fresh medium to continue culturing to obtain OCLN / CD81 / miR-122-BM-MSCs; after that, HCVcc virus was inoculated in OCLN / CD81 / miR-122-BM-MSCs to obtain the HCV cell model;

[0044] The fresh medium is DMEM / F12 medium+10%FBS+100U / mL penicillin and 100μg / mL streptomycin.

Embodiment 2

[0046] The method for establishing the HCV cell model by utilizing tree shrew bone marrow mesenchymal stem cells comprises the following steps:

[0047] First add the OCLN lentiviral vector to the tree shrew bone marrow mesenchymal stem cells, mix well and put it in 37°C, 5% CO 2 After 0.5h of infection in the incubator, add the CD81 lentiviral vector, mix well and put it in 37°C, 5% CO 2 After 0.5h of infection in the incubator, the miR-122 lentiviral vector was added at the end, mixed well and placed in 37°C, 5% CO 2 Infect in the incubator for 4 hours, then add fresh medium containing 6 μg / mL polybrene with a volume equal to 1 / 2 of the current medium volume, remove the old medium on the second day, replace with fresh medium, and continue to incubate at 37°C , 5% CO 2 Cultured in an incubator to obtain OCLN / CD81 / miR-122-BM-MSCs; finally, HCVcc virus was inoculated into OCLN / CD81 / miR-122-BM-MSCs to obtain the HCV cell model.

[0048] The fresh medium is DMEM / F12 medium+10%...

Embodiment 3

[0053] The method for establishing the HCV cell model by utilizing tree shrew bone marrow mesenchymal stem cells comprises the following steps:

[0054] Resuscitated tree shrew bone marrow mesenchymal stem cells, 5×10 5 cells / mL inoculated into a 6-well plate containing fresh medium, 2mL per well, and when the cells grew to a confluence of 50% the next day, the original medium was sucked off, and the volume added was 1 / 2 of the volume of the original medium. In fresh medium containing 6 μg / mL polybrene, according to the multiplicity of infection (MOI=15), add 7.5 μL of OCLN lentiviral vector to the tree shrew bone marrow mesenchymal stem cells, mix well at 37°C, 5% CO 2 Infect in the incubator for 1h;

[0055] Afterwards, take out the cells and add 15 μL CD81 lentiviral vector, mix well and place in 37°C, 5% CO 2 Infect in the incubator for 1h;

[0056] Finally, add 7.5 μL miR-122 lentiviral vector, mix well and place in 37°C, 5% CO 2 4 hours of infection in the incubator;...

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Abstract

The invention relates to a method for establishing an HCV (hepatitis C virus) cell model by utilizing tree shrew bone marrow mesenchymal stem cells, and belongs to the technical field of biological medicines. The method comprises the steps that firstly, an OCLN (occludin) lentiviral vector is adopted to infect the tree shrew bone marrow mesenchymal stem cells, then a CD81 lentiviral vector is added for further infection, then a miR-122 lentiviral vector is added for infection for 4 hours, then a fresh culture medium containing 4 mcg / mL of polybrene with 1 / 2 volume of a current culture medium is added, and the used culture medium is removed the next day and is replaced with the fresh culture medium for continuous culture so as to obtain the HCV (hepatitis C virus) cell model. According to the method, the cell model has the basic characteristics and the differentiation potential of the bone marrow mesenchymal stem cells and is susceptible to HCV (hepatitis C virus), HCV (hepatitis C virus) endocytosis and copy are supported, infective virus particles are produced, and new cell model resources are provided for HCV (hepatitis c virus) pathogenesis researching and drug screening.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for establishing an HCV cell model by using tree shrew bone marrow mesenchymal stem cells. Background technique [0002] Hepatitis C virus (HCV) is a virus that is seriously endangering human health. At present, the number of HCV-infected people in the world has reached 170-200 million, and the number of infected people is still increasing every year. After HCV infection, only a small number (15-25%) of infected persons clear the virus through autoimmune responses, while most (75-85%) of infected persons develop chronic infection, and 10-20% of infected persons develop liver disease. Cirrhosis, about 5% of patients with liver cirrhosis eventually develop liver cancer and die. At present, there is still no vaccine to prevent it. Although there are HCV treatment drugs, their prices are extremely expensive, and it is difficult for ordinary patients to pay. ...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10C12Q1/02
CPCG01N33/5008C12N15/86C12N2740/15043
Inventor 陆彩霞代解杰孙晓梅匡德宣王文广仝品芬李娜李明学
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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