Determination method for cigarette smoke total particle phase induced cell oxidative stress GSH/GSSG

A technology of cigarette smoke and oxidative stress, which is applied in the measurement of color/spectral characteristics, etc., can solve the problems of strict processing time, affecting measurement results, and inability to distinguish GSH and GSSG

Active Publication Date: 2015-10-28
ZHENGZHOU TOBACCO RES INST OF CNTC
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Problems solved by technology

However, this method still has some shortcomings: First, its specificity is not high enough, mainly because 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) can interact with many substances containing active sulfhydryl groups At the same time, the total amount of glutathione is ...

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  • Determination method for cigarette smoke total particle phase induced cell oxidative stress GSH/GSSG
  • Determination method for cigarette smoke total particle phase induced cell oxidative stress GSH/GSSG
  • Determination method for cigarette smoke total particle phase induced cell oxidative stress GSH/GSSG

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Embodiment Construction

[0035] The present invention is further described below in conjunction with specific examples:

[0036] The particulate phase components of cigarette smoke captured on the Cambridge filter were extracted with DMSO to prepare a sample storage solution with a concentration of total particulate matter (CSC) of 10 mg / mL, which can be stored at -80°C.

[0037] The expanded and cultured A549 cells were digested to prepare cell suspension (1×10 6 individual / mL). Add 2.0 mL of growth medium and 450 μL of cell suspension to each well of a 6-well plate, and incubate for 24 h at 37 °C and 5 % CO2. The growth medium was used to adjust the CSC stock solution to different concentrations, and the final concentration of cigarette smoke exposure was finally determined by the cytotoxicity test to be 75 μg / mL and 100 μg / mL to ensure that the cell survival rate was above 70%. After the cells were cultured for 24 h, the culture medium was aspirated, and a group of 2 wells in each column of a 6-...

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Abstract

The invention discloses a determination method for cigarette smoke total particle phase induced cell oxidative stress GSH/GSSG. The determination method comprises the following steps that 1, an experiment reagent is prepared; 2, a cigarette smoke total particle phase object is prepared; 3, cell inoculation cultivation is conducted; 4, the contamination of the cigarette smoke total particle phase object is conducted; 5, sample treatment and GSH/GSSG determination are conducted; 6, result analysis is conducted. According to the determination method for the cigarette smoke total particle phase cell induced oxidative stress GSH/GSSG, an SBB buffer solution is used for re-suspending cells, artificial oxidation GSH is prevented, and meanwhile the effective processing time of a sample is increased; in consideration of the fact that protein of active sulfhydryl exists in a cell lysis solution, in order to reduce the situation that sulfide of non-glutathione is combined with DTNB to interfere determination, before sample determination, 5-sulfosalicylic acid is added to be used for removing the protein in the sample, and interference in the determination process is reduced; since the method determines the GSH and the GSSG at the same time, the minusing is used in the result treatment, the concentrations of the GSH and the GSSG can be obtained separately, and the determination method for the cigarette smoke total particle phase cell induced oxidative stress GSH/GSSG has the advantages of being short in time-consuming, simple and convenient to operate, high in sensitivity and reliable in results.

Description

technical field [0001] The present invention relates to in vitro toxicological research on total particulate matter in cigarette smoke, specifically a method for detecting the ratio of reduced state to oxidized state of glutathione in cells after the total particulate matter in cigarette smoke induces oxidative damage (GSH / GSSG) determination method. Background technique [0002] Cigarette smoke is a complex mixture with more than 8,000 chemical components reported, and some of them show certain oxidative properties. When cells or tissues are exposed to cigarette smoke, the oxidation / antioxidation balance of the cell system is broken, the cells generate oxidative stress, and eventually lead to pathological damage in the body. When cells undergo oxidative stress, the intracellular reactive oxygen species (ROS) and reactive nitrogen species (RON) increase, causing varying degrees of oxidative damage to lipids, proteins, and DNA, while activating intracellular antioxidant sub...

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Application Information

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IPC IPC(8): G01N21/31
Inventor 李翔张世敏谢复炜赵俊伟尚平平康彧
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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