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A method for the quantitative detection of carbon monoxide-induced DNA damage by an air-liquid interface exposure system combined with high-content technology

A carbon monoxide, quantitative detection technology, applied in the field of in vitro genotoxicity measurement, can solve the problems of difficult to achieve full contact of cells, limited solubility, and changing the purpose of detection.

Active Publication Date: 2019-07-23
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

This is because some gases have limited solubility in liquid (usually water), which limits the upper limit of exposure concentration; second, some gaseous substances can react with water, which indirectly changes the purpose of detection; third, gas- Although liquid mixing exposure can make the gas dissolved in the liquid matrix fully contact with the cells for suspension cells, it is difficult to achieve sufficient contact for adherent cultured cells

Method used

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  • A method for the quantitative detection of carbon monoxide-induced DNA damage by an air-liquid interface exposure system combined with high-content technology
  • A method for the quantitative detection of carbon monoxide-induced DNA damage by an air-liquid interface exposure system combined with high-content technology
  • A method for the quantitative detection of carbon monoxide-induced DNA damage by an air-liquid interface exposure system combined with high-content technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] The induced γH2AX after 1 h of carbon monoxide exposure was measured.

[0076] Add 0.5 and 2 mL of PBS to the top and bottom of the Transwell insert, respectively, and equilibrate at 37°C for 1-2 hours. Aspirate and discard the PBS on the top and bottom of the Transwell insert, add 0.5mL to the top of the insert with a concentration of 4×10 5 The single-cell suspension of A549 cells in the logarithmic growth phase of each cell / mL was added to the bottom of the insert with 2 mL of RPMI-1640 culture medium containing 0.01moL / L HEPES, 2mmoL / LL-glutamine and 10% FBS, and incubated at 37 °C, 5% CO 2 Conditioned for 24h.

[0077] Remove the culture medium on the top of the Transwell insert, take out the top of the Transwell insert and put it into the exposure compartment of the cell exposure system, so that the permeable filter membrane in the Transwell insert and the cell exposure solution (also containing 0.01moL / LHEPES, 2mmoL / L L -Glutamine and 10% FBS RPMI-1640 culture...

Embodiment 2

[0083] The γH2AX induced by 44.64mmoL / L carbon monoxide exposure for 15, 30, 45, 60 and 90min were measured respectively.

[0084] The experiment process was carried out as described in Example 1, the only difference being that the concentration of carbon monoxide exposure was fixed at 44.64mmoL / L, and the exposure time was 0, 15, 30, 45, 60 and 90 minutes respectively.

[0085] Figure 4 Shown is the time-effect relationship curve of γH2AX produced by A549 cells induced by 44.64mmoL / L carbon monoxide at 0, 15, 30, 45, 60 and 90 minutes after exposure. It can be seen from the figure that as the exposure time increases, the γH2AX produced by A549 cells decreases compared with the normal group, and there is no significant difference in the γH2AX induced by exposure at each time point (p>0.05).

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Abstract

The invention discloses a method for quantitative detection of carbon monoxide induced cell DNA injury by adopting a gas-liquid interface exposure system combined with high content technique, which comprises the following steps: (1) cell culture; (2) carbon monoxide poisoning by gas-liquid interface exposure; (3) gamma-H2AX immunofluorescence labeling; and (4) high content detection. The method disclosed by the invention has the following advantages: high efficiency poisoning of gaseous carbon monoxide to in vitro attached cells is realized by in vitro gas-liquid interface exposure, so as to improve carbon monoxide poisoning efficiency; DNA double strand break biomarker gamma-H2AX protein produced under carbon monoxide induction is quantitatively analyzed by using automatic imaging of a high content imaging system, so that direct and rapid detection of cells is realized, and sample treatment is more convenient; and high resolution imaging not only realizes visual observation of the distribution of gamma-H2AX in nuclei, but also enables image data to be convenient for storage and re-analysis, and also can realize quantitative analysis of the gamma-H2AX induced by carbon monoxide in each cell nucleus, and the detection result is more sensitive and accurate.

Description

technical field [0001] The invention belongs to the technical field of genotoxicity determination in vitro, and more specifically, the invention relates to a method for genotoxicity determination of carbon monoxide. Background technique [0002] Carbon monoxide in the air is mainly produced by the incomplete combustion of organic matter, and the main sources are vehicle exhaust and coal combustion. Carbon monoxide can compete with oxygen for binding to hemoglobin, leading to tissue hypoxia and toxicity. However, there are few studies on the genotoxicity of carbon monoxide. Animal experiments have shown that the urine of rats exposed to carbon monoxide is not mutagenic. However, carbon monoxide may be involved in oxidative stress and DNA damage repair processes. At present, the World Health Organization Framework Convention on Tobacco Control (FCTC) lists carbon monoxide as one of the priority pollutants in smoke control. The reason why there are few studies on the genetic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/50
CPCG01N33/5014
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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