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（P<0.01），且细胞活力明显增加（P<0.05）。H2O2诱导下，pcDNA3MGST1细胞的早期凋亡率明显低于pcDNA3组\[（330±0.40）% vs （6.50±0.95）%, P<0.05\];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。

Objective:Lung adenocarcinoma SPCA1 cell line that stably overexpresses microsomal glutathione Stransferase 1 (MGST1) was constructed to explore the function and mechanism of MGST1 in lung adenocarcinoma.Methods:The recombinant plasmid pcDNA3MGST1 and the empty vector pcDNA3 were transfected into SPCA1 by Lipofectaminemediated method; after being screened by G418, stably transfected cells were labeled and divided into pcDNA3MGST1 group and empty vector pcDNA3 group. qRTPCR and Western blotting were used to detect the expression of MGST1 at mRNA and protein level in stable cells. MTS was used to detect the viability of cells. Flow cytometry and Western blotting were used to detect the cell apoptotic rate and the downstream apoptotic proteins induced by H2O2, respectively. Results: The results of sequencing showed that the recombinant plasmid pcDNA3MGST1 was successfully constructed, and the stable cell line with G418 resistance was obtained. The mRNA and protein level of MGST1 in pcDNA3MGST1 group were all significantly elevated (P<0.01), and the cell viability was significantly increased (P<0.05). Under the induction of H2O2, the early apoptosis rate of pcDNA3MGST1 group was significantly lower than that of pcDNA3 group (\[3.30±0.40\]% vs \[6.50±0.95\]%, P<0.05). The expressions of apoptotic proteins (caspase 9, caspase3 and PARP) increased, and the expressions of cleavedcaspase 9, cleavedcaspase 3 and cleavedPARP were significantly decreased in pcDNA3MGST1 group. Conclusion: The lung adenocarcinoma cell line SPCA1, which stably overexpresses human MGST1, has been successfully established. It was found that MGST1 could inhibit the apoptosis of lung adenocarcinoma cells by regulating caspase apoptosis pathway, which laid the experimental foundation for the followup study.

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）