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Determination of cellular oxidative stress gsh/gssg induced by total particulate matter in cigarette smoke

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

Active Publication Date: 2017-09-12
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 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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  • Determination of cellular oxidative stress gsh/gssg induced by total particulate matter in cigarette smoke
  • Determination of cellular oxidative stress gsh/gssg induced by total particulate matter in cigarette smoke

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[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 storage 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 ...

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Abstract

A method for determining cell oxidative stress GSH / GSSG induced by total particulate matter in cigarette smoke, comprising the following steps: 1) preparing experimental reagents; 2) preparing total particulate matter in cigarette smoke; 3) inoculating and culturing cells; 4) Exposure to total particulate matter in cigarette smoke; 5) sample processing and GSH / GSSG determination; 6) result analysis. The present invention uses SBB buffer to resuspend cells to prevent artificial oxidation of GSH and increase the effective processing time of samples; considering the active sulfhydryl protein in the cell lysate, in order to reduce the combination of non-glutathione sulfide and DTNB For interference detection, before the sample is measured, 5-sulfosalicylic acid is added to remove the protein in the sample, which reduces the interference in the measurement process; since this method detects GSH and GSSG at the same time, the subtraction method is applied in the result processing , the concentration of GSH and GSSG can be obtained respectively, and the present invention has the advantages of short time consumption, simple and convenient operation, high sensitivity and reliable 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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Patent Type & Authority Patents(China)
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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