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Detection method for vitro cell micronucleus of cigarette smoke genetic toxicity

A technology for micronucleus detection and genotoxicity is applied in the field of evaluating the genotoxicity of cigarette smoke and in vitro cell micronucleus detection of cigarette smoke genotoxicity. observation, etc.

Active Publication Date: 2010-05-19
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, each method of micronucleus detection has its disadvantages: Microscopic examination is time-consuming and laborious, and there are subjective errors; some micronuclei cannot be identified in the detection of image analysis system, resulting in false negative results; Nuclei are susceptible to release of nuclear granules from apoptotic bodies, yielding false positive results
There are many deficiencies in this method: Apoptosis of poisoned cells and apoptotic bodies appear, which interfere with micronucleus microscope observation, resulting in false positives; more than 1000 cells are counted under micronucleus microscope, and the workload is heavy; human factors may affect the micronucleus microscope count result

Method used

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  • Detection method for vitro cell micronucleus of cigarette smoke genetic toxicity
  • Detection method for vitro cell micronucleus of cigarette smoke genetic toxicity
  • Detection method for vitro cell micronucleus of cigarette smoke genetic toxicity

Examples

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Effect test

Embodiment 1

[0060] Evaluate a certain domestic flue-cured tobacco brand cigarette A. Cigarette smoke was collected through an organic phase (ethyl acetate) and an inorganic phase (deionized water). Combine the two, and adjust the concentration to 1 vial / ml with cell culture medium. Sterile filter through a 0.22 μm filter membrane and store at -80°C for later use.

[0061] Inoculate 6-well plate cells {select human bronchial epithelial cells (BEAS-2B) immortalized by adenovirus-12 / SV40 virus. At 37°C, 5% CO 2 cultured in an incubator. Cells were passaged once a week, and the medium was changed three days after passage. }, after being infected (0.002 tubes / ml, 2d), the cells were digested and collected. Take about 5 x 10 5 Place the cells in a 15ml polypropylene centrifuge tube, centrifuge (600×g, 5min), discard the supernatant, and tap to resuspend the cells. Add 300 μl of EMA staining solution, immerse in crushed ice (depth about 2cm), and visible light source (fluorescent lamp 40W)...

Embodiment 2

[0065] Evaluate a certain domestic flue-cured tobacco type cigarette B. Cigarette smoke was collected through an organic phase (ethyl acetate) and an inorganic phase (deionized water). Combine the two, and adjust the concentration to 1 vial / ml with cell culture medium. Sterile filter through a 0.22 μm filter membrane and store at -80°C for later use.

[0066] Inoculate 6-well plate cells {select human bronchial epithelial cells (BEAS-2B) immortalized by adenovirus-12 / SV40 virus. At 37°C, 5% CO 2 cultured in an incubator. Cells were passaged once a week, and the medium was changed three days after passage. }, after being infected (0.002 tubes / ml, 2d), the cells were digested and collected. Take about 5 x 10 5 Place the cells in a 15ml polypropylene centrifuge tube, centrifuge (600×g, 5min), discard the supernatant, and tap to resuspend the cells. Add 300 μl of EMA staining solution, immerse in crushed ice (about 2 cm in depth), and irradiate the cell suspension with a vis...

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Abstract

The invention relates to a detection method for vitro cell micronucleus of cigarette smoke genetic toxicity, which is characterized in that immortalized human bronchial epithelial cells are used as target cells, the smoke condensate is exposed to toxicant, the cells undergo the low-permeability and high-permeability treatment, are dyed by two fluorescent probes and analyzed by a flow cytometer. Compared with the prior art, the detection method has the following advantages of: 1.overcoming the influence of apoptotic cells on the micronucleus detection and reducing false positive results. 2. overcoming the uncertainty of the invivo micronucleus detection (animal testing) due to the adoption of the vitro cell culture; 3.adopting the six pore plate cell culture and the flow cytometer detection which can meet the high flux micronucleus detection requirements; and 4. eliminating the disturbances of chondriosome and non-specific (fragments) particles and the like by the 2500*g gradient centrifugation. Therefore, the invention improves the sensibility of the detection method and the accuracy, the objectivity and the reliability of experimental results.

Description

technical field [0001] The invention relates to the detection and analysis of the toxicity of cigarette smoke, in particular to an in vitro cell micronucleus detection method for the genotoxicity of cigarette smoke, which can be applied to evaluate the genotoxicity of cigarette smoke. Background technique [0002] Micronucleus test, as a genotoxicity detection method for inferring chromosomal damage of compounds, has the characteristics of rapidity, simplicity, and economy, and is widely used in the rapid screening of environmental compounds and the detection of a large number of occupationally exposed people (Cao Jia, Lin Zhen, Yu Zhengping. Micronucleus test ——Principle, method and its application in population monitoring and toxicity evaluation [M]. Military Medical Press, Beijing, 2000). The sensitivity, specificity, and accuracy of micronucleus detection are superior to other chromosomal aberration analysis, so many countries and international organizations stipulate it...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 谢剑平朱茂祥刘兴余杨陟华潘秀颉
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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