稳定过表达人MGST1基因抑制肺腺癌细胞SPC-A-1的凋亡

发布时间：2018-04-26 18:27

本文选题：肺腺癌 + 微粒体谷胱甘肽S转移酶　；参考：《中国肿瘤生物治疗杂志》2017年06期

【摘要】：目的:建立稳定过表达微粒体谷胱甘肽S转移酶1(microsomal glutathione S-transferase 1,MGST1)基因的肺腺癌SPC-A-1细胞系,探讨MGST1在肺腺癌中的作用及其机制。方法:重组质粒pcDNA3-MGST1和空载体pcDNA3以脂质体介导的方法转染至SPC-A-1细胞中,经过G418筛选稳转细胞系,标记为pcDNA3-MGST1细胞和空载体pcDNA3细胞。实时荧光定量PCR及Western blotting鉴定稳转细胞中MGST1 mRNA和蛋白的表达情况。MTS法检测稳转细胞的活力;流式细胞仪和Western blotting检测H_2O_2诱导下稳转细胞的凋亡率和下游凋亡相关蛋白水平变化。结果:酶切鉴定和测序结果显示pcDNA3-MGST1重组质粒构建成功,并获得具有G418抗性的稳转细胞株。pcDNA3-MGST1细胞中MGST1在mRNA和蛋白水平表达均显著升高(P0.01),且细胞活力明显增加(P0.05)。H_2O_2诱导下,pcDNA3-MGST1细胞的早期凋亡率明显低于pcDNA3组[(3.30±0.40)%vs(6.50±0.95)%,P0.05];pcDNA3-MGST1细胞凋亡相关蛋白caspase 9、caspase 3、PARP表达增多,cleaved-caspase 9、cleaved-caspase 3、cleaved-PARP表达明显减少。结论:本研究成功构建了稳定过表达MGST1的SPC-A-1肺腺癌细胞系,MGST1可能通过调节caspase凋亡通路抑制肺腺癌细胞的凋亡。
[Abstract]:Aim: to establish a lung adenocarcinoma SPC-A-1 cell line stably expressing microsomal glutathione S transferase 1(microsomal glutathione S-transferase 1 MGST1 gene, and to investigate the role of MGST1 in lung adenocarcinoma and its mechanism. Methods: recombinant plasmid pcDNA3-MGST1 and empty vector pcDNA3 were transfected into SPC-A-1 cells by liposome-mediated transfection. Stable transformed cell lines were screened by G418 and labeled as pcDNA3-MGST1 cells and empty vector pcDNA3 cells. Real-time fluorescence quantitative PCR and Western blotting were used to identify the expression of MGST1 mRNA and protein in stable transfer cells. MTS method was used to detect the activity of stable transfer cells. Flow cytometry and Western blotting were used to detect the apoptosis rate and the level of downstream apoptosis-related proteins induced by H_2O_2. Results: the results of restriction endonuclease digestion and sequencing showed that the recombinant plasmid of pcDNA3-MGST1 was successfully constructed. The expression of MGST1 at mRNA and protein levels was significantly increased in the stable transformed cell line. PcDNA3-MGST1 with G418 resistance, and the apoptosis rate of pcDNA3-MGST1 cells induced by P0.05 + H _ 2O _ 2 was significantly higher than that in pcDNA3 group [3.30 卤0.40)%vs(6.50 卤0.95p05] pcDNA3-MGST1 cells apoptosis related eggs were obtained. The expression of cleaved-caspase 3 and cleaved-caspase 3 were significantly decreased in white caspase 9, and the expression of cleaved-caspase 3 was significantly decreased. Conclusion: in this study, we successfully constructed a stable MGST1 overexpression SPC-A-1 lung adenocarcinoma cell line, MGST1, which may inhibit the apoptosis of lung adenocarcinoma cells by regulating the apoptosis pathway of caspase.
【作者单位】：南方医科大学基础医学院人体解剖国家重点学科;昆明医科大学第三附属医院暨云南省肿瘤医院肿瘤生物治疗中心;
【基金】：国家自然科学基金资助项目(No.U1502222,No.81470005,No.81260307);国家自然科学基金重大科研仪器研制项目(No.61427807)~~
【分类号】：R734.2

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