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Method for quantitatively analyzing cell DNA damage induced by benzene or benzene metabolite based on high connotation technique

A DNA damage and quantitative analysis technology, applied in the analysis of materials, material excitation analysis, material analysis by optical means, etc. It can improve the detection efficiency and sensitivity, sample processing is simple and fast, and the detection results are sensitive and accurate.

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

AI Technical Summary

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 benzene or benzene metabolite based on high connotation technique
  • Method for quantitatively analyzing cell DNA damage induced by benzene or benzene metabolite based on high connotation technique
  • Method for quantitatively analyzing cell DNA damage induced by benzene or benzene metabolite based on high connotation technique

Examples

Experimental program
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Effect test

Embodiment 1

[0066] When the metabolic activation system rat liver S9 was not added to the cell poisoning solution, the γH2AX induced after 24h exposure to benzene was measured.

[0067] 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.

[0068] Aspirate and discard the cell culture solution after culturing for 24 hours, and use cell culture solution containing 0, 2.44, 4.89, 9.77, 19.54, 39.08, 78.16, 156.25, 312.5 and 500 μg / mL benzene respectively (also containing 10% FBS and 2mmoL / L L-glutamine (RPMI-1640 culture medium) was used as the cell poisoning solution, and the cells were continued to be cultured for 24 h.

[0069] 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 ...

Embodiment 2

[0074] When the metabolic activation system rat liver S9 was added to the cell poisoning solution, the γH2AX induced after 24 hours of benzene exposure was measured.

[0075] The experimental process was carried out as described in Example 1, the only difference being that, in addition to the cell culture medium and benzene, the cell poisoning solution was mixed with 10% S9 mixture, so that the cell poisoning solution contained 1% S9 mixture.

[0076] image 3 Shown is the dose-effect relationship curve of γH2AX produced by A549 cells induced by different concentrations of benzene after 1% S9 was added to the cell poisoning solution 24 hours after exposure. It can be seen from the figure that when the concentration of benzene is below 50 μg / mL, the induced γH2AX increases with the increase of benzene concentration, and then tends to be stable with the increase of benzene concentration.

[0077] The addition of S9 to the cell poisoning solution can enhance the metabolic transfor...

Embodiment 3

[0079] When the metabolic activation system rat liver S9 was not added to the cell poisoning liquid, the γH2AX induced by hydroquinone after 24h exposure was measured.

[0080] The experimental procedure was carried out as described in Example 1, the only difference being that the cell culture solution containing 0, 0.13, 0.25, 0.51, 1.01, 2.02, 4.04, 6.06 and 8.08 μg / mL hydroquinone was used as the cell poisoning solution .

[0081] Figure 4 Shown is the dose-effect relationship curve of γH2AX induced by different concentrations of hydroquinone in A549 cells 24 hours after exposure. It can be seen from the figure that with the increase of hydroquinone concentration, the γH2AX produced by A549 cells gradually increased, showing a significant dose-effect relationship. When the concentration of hydroquinone was 6.6 μg / mL, the γH2AX produced in the cells was 1.5 times higher than that of the normal group (that is, when the concentration of hydroquinone was 0).

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Abstract

The invention discloses a method for quantitatively analyzing cell DNA damage induced by benzene or benzene metabolite 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 benzene or benzene metabolite, 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 benzene or benzene metabolite, 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, and more specifically relates to an in vitro quantitative analysis method for cellular DNA damage caused by benzene or benzene metabolites. Background technique [0002] Benzene is a common air pollutant that can induce chromosomal aberrations and chromosomal breaks in bone marrow cells. Animal experiments have shown that exposure to benzene through various routes can cause various types of tumors. At the same time, the epidemiology of occupationally exposed populations pointed out that benzene exposure may be associated with acute or chronic myeloid leukemia. At present, the International Organization for Research on Cancer (IARC) under the World Health Organization (WHO) has listed benzene as a Class 1 carcinogen, and the World Health Organization Framework Convention on Tobacco Control (FCTC) has listed benzene as one of the priority smoke control pollutants . Benze...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6486
Inventor 胡清源张森侯宏卫陈欢王安刘勇
Owner CHINA NAT TOBACCO QUALITY SUPERVISION & TEST CENT
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