香烟烟雾提取物致大鼠膈肌细胞氧化损伤

目的研究经香烟烟雾提取物（CSE）刺激后大鼠膈肌细胞8一羟基脱氧鸟苷（8-OHdG）含量的变化以及细胞内活性氧（ROS）水平和8-羟基鸟嘌呤DNA糖苷酶1（OGGl）表达对8．OHdG含量变化的影响。方法用不同浓度CSE分别刺激大鼠膈肌细胞24、48和72h后，采用高效液相色谱．电化学法（HPLC—ECD）检测大鼠膈肌细胞8．OHdG的含量，流式细胞术检测大鼠膈肌细胞ROS水平，采用实时定量PCR检~IJOGG!mRNA水平，用Western印迹法检测OGGl蛋白水平。结果经相同CSE浓度分别刺激大鼠膈肌细胞24、48和72h后，大鼠膈肌细胞中8-OHdG含量、ROS水平及OGGlmRNA和蛋白水平均无明显差异。但经不同浓度CSE刺激相同时间后，10．00％CSE和20．00％CSE刺激组大鼠膈肌细胞8-OHdG含量明显高于对照组和5．00％CSE刺激组（P〈0．05）；大鼠膈肌细胞内ROS水平、OGGlmRNA和蛋白水平较对照组均明显升高（P〈0．05）；大鼠膈肌细胞8-OHdG含量与其ROS水平呈明显正相关（r=0．826，P：0．000）；经CSE刺激后，大鼠膈肌细胞OGGlmRNA和蛋白水平均高于对照组（P〈O．05），但大鼠膈肌细胞8．OHdG含量与其OGGlmRNA和蛋白水平无明显相关（r=0．373，P=0．254；r=0．329，P=0．296）。结论CSE刺激可引起大鼠膈肌细胞8-OHdG升高，8-OHdG含量与ROS水平有关，但与其修复酶OGGl表达水平无明显相关。

Oxidative damage of diaphragm cells in rats induced by cigarette smoke extract

Objective To study the effect of cigarette smoke extract（CSE） on the diaphragm cells of rats in 8-hydroxydeoxyguanosine（8-OHdG） content and the relationship between 8-OHdG content and level of reactive oxygen species（ROS） and 8-oxoguanine DNA glycosylase-1 （OGG 1）. Methods The diaphragm cells of rats were cultured and stimulated by CSE for different time periods and at different concentrations. 8-OHdG was detected by high performance liquid chromatography with electrochemical detection（HPLC-ECD）. The level of ROS was determined by fluorescent probe of 2＇, 7＇-dichlorodihydrofluorescein diacetate （DCFH-DA）. The expression of OGG 1 mRNA was tested by real-time PCR. The expression of OGG1 protein was tested by Western blot. Results There was no significant difference in the 8-OHdG ROS, OGG1 mRNA and protein levels after CSE treatment for different time periods. However, the 8-OHdG content of the diaphragm cells induced by 10.00% CSE and 20.00% CSE was significantly increased compared with the diaphragm cells induced by 5.00% CSE and control groups（P 〈 0.05）. There was no significant difference in 8-OHdG content between 5.00% CSE and control groups（P 〉 0.05）. Compared with control, the intracellular ROS level was dramatically increased in CSE-treated groups（P 〈 0.05）, and the 8-OHdG content was increased according to the level of ROS（r = 0.826, P = 0.000）. Compared with control, the expression of OGG1 mRNA and protein were dramatically increased in CSE-treated groups（P 〈 0.05）, however, the 8-OHdG content has but not with the expression of OGG1 mRNA and protein（r = 0.373, P = 0.254; r = 0.329, P = 0.296）. Conclusions CSE could increase the level of 8-OHdG in the diaphragm cells of rats. The 8-OHdG content has correlation with ROS level, but not with the expression of OGG 1.