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目的探讨长期缺氧对 SPCA1细胞谷胱甘肽转移酶(GST-π)表达的影响及耐药性的改变。方法 SPCA1细胞常氧(20%O_2)和缺氧(0.5%O_2)条件下作用16 h 后,提取 RNA 和蛋白,用定量 RT-PCR 的方法检测缺氧诱导因子1α(HIF-1α)和GST-π mRNA 的表达情况,用 Western blotting法检测 HIF-1α蛋白;制备细胞悬液,用流式细胞仪测定 GST-π表达;通过克隆形成实验分析 SPCA1细胞对阿霉素和丝裂霉素的药物敏感性。通过 RNA 干扰技术下调 HIF-1α表达后,分为对照组和干扰组,观察 GST-π的表达。结果设常氧组 GST-π mRNA 表达率为1,缺氧组为12.2。常氧组 GST-π蛋白阳性表达率为(35.78±1.25)%,缺氧组为(72.13±2.11)%。与常氧组比较缺氧组 HIF-1α表达明显增加。计算1%克隆存活的药物浓度,阿霉素处理细胞,常氧组浓度为(0.29±0.05)μg/ml;缺氧组浓度为(0.48±0.07)μg/ml;丝裂霉素处理细胞,常氧组浓度为(0.71±0.10)μg/ml;缺氧组浓度为(0.79±0.12)μg/ml。干扰 HIF-1α后,HIF-1α mRNA 与对照组比较下降了70%,而 GST-π mRNA稍有下降。GST-π蛋白表达常氧条件下对照组为(32.14±1.23)%,干扰组为(30.88±1.65)%;缺氧条件下对照组为(64.78±2.45)%,干扰组为(67.42±2.21)%。结论长期缺氧可诱导 SPCA1细胞HIF-1α和 GST-π表达增加,对阿霉素耐药性增加,对丝裂霉素没有影响,HIF-1α和 GST-π并没有直接的相关性。
Objective To investigate the effect of chronic hypoxia on the expression of glutathione transferase (GST-π) and the change of drug resistance in SPCA1 cells. Methods RNA and protein were extracted from SPCA1 cells under normoxia (20% O 2) and hypoxia (0.5% O 2) for 16 h, and the expressions of HIF-1α and GST were detected by quantitative RT- -π mRNA expression was detected by Western blotting assay of HIF-1α protein; Preparation of cell suspension, GST-π expression was measured by flow cytometry; Analysis of the clonal formation of SPCA1 cells to doxorubicin and mitomycin Drug sensitivity. After down-regulating the expression of HIF-1α by RNAi technology, the cells were divided into control group and interference group to observe the expression of GST-π. Results The normoxic group GST-π mRNA expression rate of 1, hypoxia group was 12.2. The positive expression rate of GST-π protein in normoxia group was (35.78 ± 1.25)%, (72.13 ± 2.11)% in hypoxia group. Compared with normoxia group, the expression of HIF-1α in hypoxia group increased significantly. The drug concentration of 1% clone was calculated. The cells treated with doxorubicin had a concentration of (0.29 ± 0.05) μg / ml in the normoxia group and (0.48 ± 0.07) μg / ml in the hypoxia group. The normoxia group was (0.71 ± 0.10) μg / ml and the hypoxia group was (0.79 ± 0.12) μg / ml. After interference with HIF-1α, HIF-1α mRNA decreased by 70% compared with the control group, while GST-π mRNA decreased slightly. The expression of GST-π protein was (32.14 ± 1.23)% in the control group and (30.88 ± 1.65)% in the normoxia group, 64.78 ± 2.45% in the control group and 67.42 ± 2.21 )%. CONCLUSION: Long-term hypoxia can induce the expression of HIF-1α and GST-π in SPCA1 cells to increase, and the drug resistance to doxorubicin increases without affecting mitomycin. There is no direct correlation between HIF-1α and GST-π.