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

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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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 detected, and GSH and GSSG cannot be distinguished; in addition, this method has strict requirements on the sample processing time, and the time from the beginning of sample preparation to detection should not exceed 3 minutes, otherwise the determination will be affected result

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