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目的:探讨人肺腺癌细胞株A549及紫杉醇(paclitaxel)耐药细胞株A549/Taxol对于H2O2氧化刺激的不同反应性及可能的作用机制。方法:用不同浓度的H2O2处理A549及A549/Taxol细胞后,采用细胞计数试剂盒-8(cell counting kit-8,CCK-8)检测细胞存活率,并计算半数细胞抑制浓度(half inhibitory concentration,IC50)值;采用荧光探针氯甲基-2’,7’-二氯二氢荧光素二乙酯[5-(and-6)-chloromethyl-2’,7’-dichlorouorescein diacetateacctyl ester,CM-H2DCFDA]染色法检测细胞内活性氧(reactive oxygen species,ROS)水平;四甲基罗丹明甲酯(tetramethylrhodamine methyl ester perchlorate,TMRM)染色法检测细胞线粒体膜电位水平;AnnexinⅤ-FITC/PI双染法检测细胞凋亡率;实时荧光定量-PCR法检测细胞内超氧化物歧化酶1(superoxide dismutase1,SOD1)、SOD2、过氧化氢酶(catalase,CAT)、γ-谷氨酰半胱氨酸链接酶(glutamate-cysteineligase,GCL)、谷胱甘肽合成酶(glutathione synthetase,GSS)和谷胱甘肽还原酶(glutathione reductase,GSR)mRNA表达的变化。结果:耐药细胞株A549/Taxol对H2O2的IC50值高于亲本细胞株A549;采用低毒浓度H2O2(50和100μmol/L)作用2h后,A549/Taxol细胞中的ROS水平增高幅度显著低于A549细胞,A549/Taxol细胞的线粒体膜电位保持相对稳定而A549细胞显著增高,且A549/Taxol细胞的凋亡发生率显著低于A549细胞。A549/Taxol细胞中抗氧化系统相关基因SOD1、SOD2、CAT、GCL、GSS和GSRmRNA的表达量高于A549细胞;在H2O2作用后,A549细胞中上述基因表达量显著增高,而A549/Taxol细胞中仅GSRmRNA表达上调。结论:紫杉醇耐药细胞对氧化刺激的耐受能力显著高于敏感细胞株,其机制可能与耐药细胞自身氧化-还原平衡系统的适应性变化有关。
OBJECTIVE: To investigate the different reactivity and possible mechanism of human lung adenocarcinoma cell line A549 and paclitaxel-resistant A549 / Taxol on H2O2 oxidation. Methods: A549 and A549 / Taxol cells were treated with different concentrations of H2O2, cell viability was measured by cell counting kit-8 (CCK-8), and the half inhibitory concentration (IC50) was determined by fluorescence spectroscopy. Fluorescent probe was used to detect the cytotoxicity of CM-2, 7-dichlorouorescein diacetateacctyl ester and CM- H2DCFDA] staining was used to detect the levels of reactive oxygen species (ROS) in the cells; mitochondrial membrane potential (MTP) was measured by TMRM staining; AnnexinⅤ-FITC / PI double staining The apoptosis rate of the cells was detected by real-time fluorescence quantitative-PCR. The expressions of superoxide dismutase 1 (SOD1), superoxide dismutase 1 (SOD1), catalase (CAT) and γ-glutamylcysteine Changes of mRNA expression of glutamate-cysteineligase (GCL), glutathione synthetase (GSS) and glutathione reductase (GSR) Results: The IC50 value of A549 / Taxol against H2O2 was higher than that of the parental cell line A549. The increase of ROS level in A549 / Taxol cells was significantly lower than that of A549 cells after treated with low concentration of H2O2 (50 and 100μmol / L) for 2h A549 cells, A549 / Taxol cells mitochondrial membrane potential remained relatively stable and A549 cells was significantly increased, and A549 / Taxol cells apoptosis was significantly lower than A549 cells. The expression of SOD1, SOD2, CAT, GCL, GSS and GSR mRNA in A549 / Taxol cells was higher than that in A549 cells. The expression of these genes was significantly increased in A549 / Taxol cells after A549 / Taxol treatment Only GSR mRNA expression is up-regulated. Conclusion: The tolerance of paclitaxel-resistant cells to oxidative stress is significantly higher than that of the sensitive cell lines, and the mechanism may be related to the adaptive changes of self-oxidation-reduction equilibrium system of drug-resistant cells.