Method for quantitatively analyzing cell DNA damage induced by benzo[a]pyrene based on high connotation technique

A quantitative analysis, cell technology, applied in the analysis of materials, material excitation analysis, material analysis through optical means, etc., can solve the problems of destroying cell structure and functional integrity, low detection throughput, cumbersome operation, etc., and achieve sample processing Simple and fast, sensitive and accurate detection results, high detection throughput

Inactive Publication Date: 2017-08-18
CHINA NAT TOBACCO QUALITY SUPERVISION & TEST CENT +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Flow cytometry and Western Blot are cumbersome to operate, and adherent cells need to be enzymatically hydrolyzed into single-cell suspension before detection, which destroys the structural and functional integrity of cells, and the detection throughput is low
ELISA cannot provide the distribution of fluorescent focal points in cells and needs to add other detection proteins to correct the results, while the detection throughput of microscopy technology is low, and the error of human counting is large

Method used

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  • Method for quantitatively analyzing cell DNA damage induced by benzo[a]pyrene based on high connotation technique
  • Method for quantitatively analyzing cell DNA damage induced by benzo[a]pyrene based on high connotation technique
  • Method for quantitatively analyzing cell DNA damage induced by benzo[a]pyrene based on high connotation technique

Examples

Experimental program
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Effect test

Embodiment 1

[0064] The γH2AX induced by benzo[a]pyrene after 24h exposure was measured when the metabolic activation system rat liver S9 was not added to the cell poisoning solution.

[0065] A549 cells in logarithmic growth phase were collected, planted in 96-well plates at 10,000 cells per well, in RPMI-1640 medium containing 10% FBS and 2mmoL / L L-glutamine at 37°C, 5% CO 2 Incubate in the incubator for 24h.

[0066] Aspirate and discard the cell culture fluid after culturing for 24 h, and use cell culture fluid containing 0, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 μmol / L benzo[a]pyrene (also containing 10% FBS and 2mmoL / L L-glutamine RPMI-1640 culture solution) was used as the cell poisoning solution, and the cells were continued to be cultured for 24h.

[0067] After the poisoning, discard the cell poisoning solution, add 100 μL PBS to each well and wash twice for at least 5 minutes each time; then add 50 μL 4% paraformaldehyde solution to each well and fix at room temperature f...

Embodiment 2

[0072] The γH2AX induced by benzo[a]pyrene after 24h exposure was measured when rat liver S9, a metabolic activation system, was added to the cell poisoning solution.

[0073] The experimental procedure was carried out as described in Example 1, the only difference being that, in addition to the cell culture medium and benzo[a]pyrene, the cell poisoning solution was mixed with a 10% S9 mixture, so that the cell poisoning solution contained 1% of the S9 mixture .

[0074] image 3 Shown is the dose-effect relationship curve of γH2AX produced by A549 cells induced by different concentrations of benzo[a]pyrene after 1% S9 was added to the cell poisoning solution 24 hours after the poisoning. As can be seen from the figure, with the increase of the exposure concentration, the γH2AX produced in the cells continued to increase, showing a significant dose-effect relationship. When the concentration of benzo[a]pyrene exceeded 25 μmoL / L, the γH2AX induced by it was 1.5 times of the n...

Embodiment 3

[0077] When the metabolic activation system rat liver S9 was not added to the cell poisoning solution, the γH2AX induced by 25 μmoL / L benzo[a]pyrene after exposure for 1, 2, 4, 8, 12 and 24 hours were measured.

[0078]The experimental process was carried out as described in Example 1. The only difference was that the concentration of benzo[a]pyrene in the cell exposure solution was fixed at a concentration of 25 μmoL / L, and the exposure time was 1, 2, 4, 8, 12 and 24h.

[0079] Figure 4 Shown is the time-effect relationship curve of γH2AX produced by A549 cells induced by 25 μmoL / L benzo[a]pyrene at 1, 2, 4, 8, 12 and 24 hours after exposure. It can be seen from the figure that as the exposure time increases, the γH2AX produced by A549 cells gradually increases, reaches the maximum at 12h, and then tends to be stable.

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Abstract

The invention discloses a method for quantitatively analyzing cell DNA damage induced by benzo[a]pyrene based on a high connotation technique. The method comprises the following steps: 1) performing cell culture; 2) infecting cells; 3) performing immunofluorescent labeling for gamma H2AX; and 4) performing quantitative analysis by adopting the high connotation technique. The method has the advantages that a high connotation imaging system is utilized for automatically imaging and quantitatively analyzing the gamma H2AX protein of a DNA double-strand breaking mark induced by benzo[a]pyrene, the direct and quick detection for cells is realized, and the sample treatment is more convenient; and due to high-resolution imaging, the distribution of gamma H2AX in a cell nucleus can be directly observed, the image data can be conveniently stored and analyzed, and the quantitative analysis for gamma H2AX, induced in each cell nucleus, of benzo[a]pyrene, can be realized, so that the detection result is more sensitive and accurate.

Description

technical field [0001] The invention belongs to the technical field of in vitro detection of DNA damage, more specifically, the invention relates to an in vitro quantitative analysis method for cellular DNA damage caused by benzo[a]pyrene. Background technique [0002] Benzo[a]pyrene is a typical representative of polycyclic aromatic hydrocarbons, and a lot of evidence supports that benzo[a]pyrene is an important cause of lung cancer and other cancers in smokers. At present, the International Organization for Research on Cancer (IARC) under the World Health Organization (WHO) has listed benzo[a]pyrene as a class 1 carcinogen, and the World Health Organization Framework Convention on Tobacco Control (FCTC) has listed benzo[a]pyrene Listed as one of the flue gas priority control pollutants. Benzo[a]pyrene can form adducts with DNA, leading to mutations in the tumor suppressor gene p53 and oncogenes. In addition, benzo[a]pyrene can also induce DNA strand breaks, chromosomal a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6486
Inventor 侯宏卫张森胡清源陈欢王安刘勇
Owner CHINA NAT TOBACCO QUALITY SUPERVISION & TEST CENT
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