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目的:探讨Smac基因过表达对食管癌细胞株Eca109顺铂化疗敏感性的影响。方法:脂质体介导将带有GFP的pcDNA3.1-Smac重组体及带有GFP的pcDNA3.1空白载体转染入食管癌细胞株Eca-109,G418筛选阳性克隆,荧光显微镜下观察转染效率,Western blot检测转染前后细胞内Smac蛋白表达水平的变化。顺铂处理组(1、5、10 mg/L)和顺铂未处理组分别处理转染前后的食管癌细胞株Eca109,Annexin V/PI双染法经流式细胞术检测细胞凋亡率。结果:分别建立稳定表达Smac基因+GFP基因,新霉素抗性基因(neo)+GFP基因的食管癌亚克隆细胞株Eca109/Smac,Eca109/neo。Eca109/Smac的Smac蛋白表达水平明显高于Eca109/neo和Eca109(P<0.05)。在顺铂未处理组,Eca109/Smac、Eca109/neo和Eca109的细胞凋亡率组间无明显统计学差异。在顺铂处理组,顺铂浓度分别为1 mg/L、5 mg/L、10 mg/L,Eca109/Smac的细胞凋亡率明显高于Eca109/neo和Eca109,组间具有统计学差异(P<0.05),且随着顺铂浓度的升高,Eca109/Smac细胞凋亡率随之升高(P<0.05)。Eca109/neo和Eca109组间无明显统计学差异。结论:Smac基因转染未经顺铂处理的Eca109细胞株中不诱发凋亡,在顺铂处理组其过表达能增强Eca109对顺铂的化疗敏感性。
Objective: To investigate the effect of Smac gene overexpression on chemosensitivity of cisplatin in esophageal carcinoma cell line Eca109. Methods: The pcDNA3.1-Smac recombinant with GFP and pcDNA3.1 with GFP were transfected into esophageal cancer cell line Eca-109 by lipofectamine. The positive clones were screened by G418 and observed under fluorescence microscope Staining efficiency, Western blot detection of intracellular Smac protein expression before and after transfection. Cisplatin treated group (1, 5, 10 mg / L) and Cisplatin untreated group were treated with Eca109 before and after transfection. The apoptosis rate was detected by flow cytometry with Annexin V / PI double staining. Results: The esophageal subclone cell lines Eca109 / Smac and Eca109 / neo were established respectively, which express Smac gene + GFP gene and neo + GFP gene. Smac protein expression of Eca109 / Smac was significantly higher than that of Eca109 / neo and Eca109 (P <0.05). There was no significant difference in apoptosis rates of Eca109 / Smac, Eca109 / neo and Eca109 in cisplatin untreated group. The apoptosis rate of Eca109 / Smac in cisplatin-treated group was significantly higher than that of Eca109 / neo and Eca109 at cisplatin concentrations of 1 mg / L, 5 mg / L and 10 mg / L, respectively P <0.05). With the increase of cisplatin concentration, the apoptosis rate of Eca109 / Smac cells increased (P <0.05). Eca109 / neo and Eca109 no significant statistical difference between groups. CONCLUSION: Smac gene transfection does not induce apoptosis in Eca109 cells without cisplatin treatment. Overexpression of Eca109 in cisplatin-treated group can enhance chemosensitivity of Eca109 to cisplatin.