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Method for quantitatively detecting 1,3-butadiene-induced cell DNA damage in combination with gas-liquid interface exposure system and high-content technology

A quantitative detection and liquid interface technology, applied in the field of genotoxicity determination in vitro, can solve the problems of changing the purpose of detection, difficult to achieve full contact of cells, limited solubility, etc.

Active Publication Date: 2017-09-01
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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  • Method for quantitatively detecting 1,3-butadiene-induced cell DNA damage in combination with gas-liquid interface exposure system and high-content technology
  • Method for quantitatively detecting 1,3-butadiene-induced cell DNA damage in combination with gas-liquid interface exposure system and high-content technology
  • Method for quantitatively detecting 1,3-butadiene-induced cell DNA damage in combination with gas-liquid interface exposure system and high-content technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] When the metabolic activation system rat liver S9 was not added to the cell poisoning solution, the γH2AX induced by 1,3-butadiene exposure for 1 h was measured.

[0079] Add 0.5 and 2 mL of PBS to the top and bottom of the Transwell plug-in, respectively, and equilibrate for 1-2 hours at 37°C. Aspirate and discard the PBS on the top and bottom of the Transwell plug-in, and add 0.5 mL to the top of the plug-in to a concentration of 4×10 5 A single cell suspension of A549 cells in the logarithmic growth phase was added to the bottom of the plug-in unit with 2 mL of RPMI-1640 medium containing 0.01moL / L HEPES, 2mmoL / LL-glutamine and 10% FBS, and ℃, 5% CO 2 Culture for 24h under conditions.

[0080] Remove the culture solution on the top of the Transwell plug-in, take out the top of the Transwell plug-in and put it into the poisoning chamber of the cell exposure system, so that the permeable filter membrane and the cell staining solution in the Transwell plug-in (also contain 0...

Embodiment 2

[0086] When the metabolic activation system rat liver S9 was added to the cell poisoning solution, the γH2AX induced by 1,3-butadiene exposure for 1 h was measured.

[0087] The experiment process was carried out as described in Example 1. The only difference was that the cell poisoning solution in the poisoning chamber was a mixture of cell culture fluid and 10% S9 mixture, so that the cell poisoning solution contained 1% S9 mixture.

[0088] Figure 4 Shown is the dose-effect relationship curve of γH2AX produced by A549 cells with different concentrations of 1,3-butadiene after adding 1% S9 to the poisoned culture solution, and image 3 The results shown are basically the same.

[0089] The addition of S9 to the cell venom can enhance the metabolic transformation of 1,3-butadiene in vitro. Since 1,3-butadiene is a pre-genotoxic substance, the addition of the in vitro metabolic activation system S9 can avoid the possibility of false negative test results due to insufficient cell met...

Embodiment 3

[0091] When the metabolic activation system rat liver S9 was not added to the cell poisoning solution, the γH2AX induced by 35.18mmoL / L 1,3-butadiene was measured after exposure to 15, 30, 45, 60 and 90 minutes.

[0092] The experiment process was carried out as described in Example 1. The only difference is that the concentration of 1,3-butadiene was fixed at a concentration of 35.18mmoL / L, and the exposure time was 0, 15, 30, 45, 60, and 90 min. .

[0093] Figure 5 Shown is the time-effect relationship curve of 35.18mmoL / L 1,3-butadiene inducing γH2AX produced by A549 cells 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 gradually increases, showing a significant time-effect relationship. When the exposure time exceeds 60 minutes, the induced γH2AX is more than 1.5 times that of the normal group.

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Abstract

The invention discloses a method for quantitatively detecting 1,3-butadiene-induced cell DNA damage in combination with a gas-liquid interface exposure system and a high-content technology. The method comprises the following steps: 1) cell culture; 2) 1,3-butadiene exposure through a gas-liquid interface exposure mode; 3) immunofluorescent labeling of gamma H2AX; 4) high-content detection. The method disclosed by the invention has the advantages that the efficient exposure of gaseous 1,3-butadiene to wall-attached cells in vivo is realized by adopting the in-vitro gas-liquid interface exposure mode, so that the exposure efficiency of the 1,3-butadiene is improved; a high-content imaging system is utilized for automatic imaging, 1,3-butadiene-induced DNA double-chain fracture maker gamma H2AX proteins are quantitatively analyzed, and the direct and quick detection of the cells is realized, so that the sample treatment is more convenient; through high-resolution imaging, not only can the distribution of the gamma H2AX in cell nucleuses be directly observed, but also image data is convenient to store and re-analyze, and the quantitative analysis on the 1,3-butadiene-induced gamma H2AX in each of the cell nucleuses can be realized, so that detection results are more sensitive and accurate.

Description

Technical field [0001] The present invention belongs to the technical field of in vitro genotoxicity determination. More specifically, the present invention relates to a method for determining the genotoxicity of 1,3-butadiene. Background technique [0002] 1,3-butadiene is a common atmospheric and environmental pollutant that can induce multiple organ tumors through inhalation. In addition, 1,3-butadiene epoxy metabolites can form DNA adducts and induce Hprt (hypoxanthine phosphoribosyl transferase) gene mutations. At present, the International Organization for Research on Cancer (IARC) under the World Health Organization (WHO) has classified 1,3-butadiene as a category 1 carcinogen, and the World Health Organization Framework Convention on Tobacco Control (FCTC) has classified 1,3-butadiene Alkenes are listed as one of the priority control pollutants of flue gas. Therefore, the accurate detection of 1,3-butadiene gas's damage to cell DNA is of great significance for 1,3-butad...

Claims

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Application Information

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IPC IPC(8): G01N33/58G01N33/50
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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