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A method for establishing hcv cell model using tree shrew bone marrow mesenchymal stem cells

A cell model, bone marrow mesenchymal technology, applied in the field of biomedicine, can solve the problem of hepatocytes cannot be passaged

Active Publication Date: 2022-07-19
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

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  • A method for establishing hcv cell model using tree shrew bone marrow mesenchymal stem cells
  • A method for establishing hcv cell model using tree shrew bone marrow mesenchymal stem cells
  • A method for establishing hcv cell model using tree shrew bone marrow mesenchymal stem cells

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Effect test

Embodiment 1

[0042] The method for establishing an HCV cell model using tree shrew bone marrow mesenchymal stem cells includes 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. The fresh medium containing 2 μg / mL polybrene was 1 / 2 of the base volume. On the second day, the old medium was removed and replaced with fresh medium to continue culturing to obtain OCLN / CD81 / miR-122-BM-MSCs; after that, the HCVcc virus was inoculated into OCLN / CD81 / miR-122-BM-MSCs to obtain HCV cell model;

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

Embodiment 2

[0046] The method for establishing an HCV cell model using tree shrew bone marrow mesenchymal stem cells includes the following steps:

[0047] First add OCLN lentiviral vector to tree shrew bone marrow mesenchymal stem cells, mix well and put it at 37°C, 5% CO 2 After 0.5 hours of infection in the incubator, the CD81 lentiviral vector was added, mixed and placed at 37°C, 5% CO. 2 After 0.5 h of infection in the incubator, the miR-122 lentiviral vector was added at the end, and after mixing, put it at 37°C, 5% CO. 2 Infection in the incubator for 4 hours, after which fresh medium containing 6 μg / mL polybrene was added in a volume of 1 / 2 of the current medium volume. The next day, the old medium was removed, replaced with fresh medium, and the temperature was continued at 37°C. , 5% CO 2 Cultivated in an incubator to obtain OCLN / CD81 / miR-122-BM-MSCs; finally, inoculate HCVcc virus into OCLN / CD81 / miR-122-BM-MSCs to obtain an HCV cell model.

[0048] The fresh medium was DMEM / ...

Embodiment 3

[0053] The method for establishing an HCV cell model using tree shrew bone marrow mesenchymal stem cells includes the following steps:

[0054] Resuscitate tree shrew bone marrow mesenchymal stem cells, press 5 × 10 5 Cells / mL were inoculated into 6-well plates containing fresh medium, 2 mL per well. When the cells grew to 50% confluence the next day, the original medium was aspirated, and the added volume was 1 / 2 of the original medium volume. In fresh medium containing 6μg / mL polybrene, according to the multiplicity of infection MOI=15, 7.5μL of OCLN lentiviral vector was added to tree shrew bone marrow mesenchymal stem cells, and after mixing, 37°C, 5% CO 2 Infection in the incubator for 1h;

[0055] After that, the cells were taken out and 15 μL of CD81 lentiviral vector was added, mixed and put into 37°C, 5% CO. 2 Infection in the incubator for 1h;

[0056] Finally, 7.5 μL of miR-122 lentiviral vector was added, and after mixing, put it at 37°C and 5% CO. 2 4 hours of...

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Abstract

The invention relates to a method for establishing an HCV cell model by utilizing tree shrew bone marrow mesenchymal stem cells, and belongs to the technical field of biomedicine. In the method, the tree shrew bone marrow mesenchymal stem cells are infected with the OCLN lentiviral vector, then the CD81 lentiviral vector is added to continue the infection, and then the miR-122 lentiviral vector is added for infection. After 4 hours of infection, the volume of The fresh medium containing 4 μg / mL polybrene was 1 / 2 of the current medium volume. On the second day, the old medium was removed and replaced with fresh medium to continue culturing to obtain the HCV cell model, which has the characteristics of bone marrow mesenchymal stem cells. Basic characteristics and differentiation potential, susceptibility to HCV, support HCV entry, replication and production of infectious virus particles, providing new cell model resources for the study of HCV pathogenic mechanism and drug screening.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and particularly relates to a method for establishing an HCV cell model by utilizing tree shrew bone marrow mesenchymal stem cells. Background technique [0002] Hepatitis C virus (HCV) is a virus that is seriously endangering human health. Currently, there are 170-200 million people infected with HCV in the world, and the number of infected people is still increasing every year. Following HCV infection, only a minority (15-25%) of infected individuals clear the virus through an autoimmune response, while the majority (75-85%) develop chronic infection, of which 10-20% develop liver disease Cirrhosis, about 5% of patients with cirrhosis eventually develop liver cancer until death. There is still no vaccine to prevent it, and although HCV treatment drugs are available, they are extremely expensive and difficult for ordinary patients to pay. Appropriate cell models and animal models are neede...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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