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Method for analyzing tar-induced cell DNA (deoxyribonucleic acid) damage on basis of quantitative analysis of high content technology

A DNA damage and quantitative analysis technology, applied in the analysis of materials, material excitation analysis, material analysis by optical means, etc., can solve the problem of unable to provide intracellular fluorescence focus distribution, damage to cell structure and functional integrity, microscopy technology detection problems such as low throughput, to achieve the effect of simple and fast sample processing, improved detection efficiency and sensitivity, and sensitive and accurate detection results

Inactive Publication Date: 2017-07-21
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 analyzing tar-induced cell DNA (deoxyribonucleic acid) damage on basis of quantitative analysis of high content technology
  • Method for analyzing tar-induced cell DNA (deoxyribonucleic acid) damage on basis of quantitative analysis of high content technology
  • Method for analyzing tar-induced cell DNA (deoxyribonucleic acid) damage on basis of quantitative analysis of high content technology

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0063] Preparation of tar: standard cigarette sample 3R4F was equilibrated at temperature (22±1)°C and relative humidity 60%±3% for 48 hours, then smoked the cigarette with a rotary smoking machine, collected the total particle phase with a Cambridge filter, and then Add a certain amount of DMSO (dimethyl sulfoxide) to prepare a smoke particulate matter solution (tar) with a concentration of 100 mg / mL, and store it at -80°C for future use.

[0064] A high-content cell analysis system was purchased from Thermo Scientific, model ArrayScan VTI600.

Embodiment 1

[0066] When the metabolic activation system rat liver S9 was not added to the cell poisoning liquid, the induced γH2AX after tar exposure for 24 hours 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] The cell culture fluid after culturing for 24 h was discarded, and the cell culture fluid (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 hours.

[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 fix at room temperature for 15 minutes; fix the cells with PBS buffer Wash twice for at least 5min each time; then...

Embodiment 2

[0074] When the metabolic activation system rat liver S9 was added to the cell poisoning solution, the γH2AX induced after tar exposure for 24 hours 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 tar, 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 tar 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 gradually increased, showing a significant dose-effect relationship. When the tar concentration was 250μg / mL, the γH2AX produced in the cells was 1.5 times higher than that of the normal group.

[0077] The addition ...

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Abstract

The invention discloses a method for analyzing tar-induced cell DNA (deoxyribonucleic acid) damage on the basis of quantitative analysis of the high content technology. The method includes the steps of 1), cell culture; 2), cell contamination; 3), immunofluorescent labeling of gamma H2AX; 4), quantitative analysis of high content technology. The method has the advantages that automatic imaging is performed by the aid of a high content imaging system, tar-induced gamma H2AX protein of DNA double-strand fracture markers is quantitatively analyzed, cells are detected directly and rapidly, and sample processing is facilitated; distribution of gamma H2AX in cell nucleuses can be observed directly and image materials are stored and reanalyzed conveniently through high-resolution imaging, the tar-induced gamma H2AX in each cell nucleus can be quantitatively analyzed, and detection results are 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 tar-induced cellular DNA damage. Background technique [0002] Cigarette smoke can be divided into two phases according to the state of matter, one is the gas phase, and the other is the granular phase, and the granular phase material is tar. Cigarette tar contains a variety of toxic substances, such as superoxide anion O· - , hydrogen peroxide and other reactive oxygen species, aldehydes, benzopyrene, etc. Tar can induce a variety of cancers and respiratory diseases, and the active free radicals and toxic substances in it can induce oxidative damage to DNA, DNA strand breaks, micronuclei, gene mutations, etc. Therefore, accurate detection of tar damage to cellular DNA is of great significance for the genotoxicity and hazard evaluation of tar. [0003] DNA double-strand break is the most seri...

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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