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Method for detecting cytotoxicity of anti-HCV medicament

A cytotoxicity, cell technology, used in biochemical equipment and methods, determination/inspection of microorganisms, measurement of color/spectral properties, etc.

Inactive Publication Date: 2010-07-14
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, there is no report on the method of detecting the cytotoxicity of anti-HCV drugs by MTT method based on HCV cell culture system (HCVcc) in the prior art

Method used

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  • Method for detecting cytotoxicity of anti-HCV medicament
  • Method for detecting cytotoxicity of anti-HCV medicament
  • Method for detecting cytotoxicity of anti-HCV medicament

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] 1. Huh 7.5.1 cell growth curve

[0030] The cell growth curve was measured by MTT method to determine the experimental cell density.

[0031] (1) Take the Huh 7.5.1 cells in the logarithmic growth phase, digest with 0.25% trypsin, centrifuge at 800-1000rpm / mim for 3min, count on the hemocytometer, and adjust the cell concentration to 9×10 4 cells / ml, spread in 96-well plate (100μl / well).

[0032] (2) Supplement the medium to a final volume of 200 μl / well, and place the culture plate at 37°C with 5% CO 2 For culture, supplement 50-100 μl fresh medium every three days. Set up 3 repeated holes, and set up a blank zeroing group at the same time.

[0033] (3) Add 20μl 5mg / ml MTT solution to the experimental well every day, 37℃5%CO 2 After incubating in the incubator for 4 hours, discard the supernatant, add 150 μl DMSO, shake and dissolve for 10 minutes, measure the OD of each well with a microplate reader 490 value.

[0034] (4) Draw the growth curve of Huh 7.5.1 cell...

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Abstract

The invention relates to a method for detecting cytotoxicity of an anti-HCV medicament. 2a-type chimeric virus J6 / JFH1 is utilized to transfect Huh7.5.1 cell to generate an HCV cell culture system (HCV cell culture, HCVcc) with infective virus particles, an MTT method for analyzing cell activity and cell growth and proliferation is utilized to detect the cytotoxicity of the HCV medicament, the inhibition ratio of the medicament to cell growth is calculated, and IC50 is obtained by calculation through Graphpad Prism 5. The method has the advantage of simple operation, economy, rapidness, sensitivity and no radioactive pollution, can accurately detect the cytotoxicity of the medicament, has good repeatability, and is an accurate detection method for smoothly implementing evaluation of in-vitro antiviral medicinal effect.

Description

technical field [0001] The invention belongs to the field of antiviral drug screening, and in particular relates to a method for detecting cytotoxicity of anti-HCV drugs. Background technique [0002] It is estimated that about 170 million people worldwide are infected with HCV. Persistent infection and chronic liver disease occur in about 80% of cases of initial HCV infection. Although chronic HCV infection can remain asymptomatic, it can also cause severe liver damage and eventually develop. become cirrhosis and liver cancer. Currently, there is no effective vaccine against HCV. Interferon-α (IFN-α) combined with ribavirin is used in clinical treatment, and the virological sustained response reaches 50%, but long-term use of ribavirin will lead to hemolytic Adverse reactions such as anemia, so the development of new therapeutic drugs is of great significance. The screening systems for anti-HCV drugs mainly include: the replicon system, the HCV genome that can replicate a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02G01N21/31
Inventor 夏雪山唐颖蕾冯悦魏大巧刘丽
Owner KUNMING UNIV OF SCI & TECH
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