Smac基因过表达增强顺铂对食管癌Eca109细胞化疗的敏感性

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。

Overexpression of Smac gene enhanced chemotherapeutic sensitivity of esophageal cancer cell line Eca109 to cisplatin

AIM: To investigate the effects of Smac gene overexpression on chemotherapeutic sensitivity of esophageal cancer cell line Eca109 to cisplatin.METHODS: pcDNA3.1-Smac with GFP and pcDNA3.1-Smac with GFP were transfected into esophageal cancer cell line Eca109 by liposome and incubated with G418 for subclone selection.The efficiency of transfection was observed under fluorescence microscope,cellular Smac gene expression were determined by Western blot.Cisplatin treated group(1,5,10 mg/L) and cisplatin untreated group were selected to treat untransfected and transfected esophageal cancer cell line Eca109.Apoptosis was determined by Annexin V/PI.RESULTS: The subclone esophageal cancer cell line Eca109,stable expressing Smac+GFP and neo+GFP respectively,were successfully selected,named as Eca109/Smac,Eca109/neo.Compared with the Eca109/neo and Eca109,the Smac expression level of Eca109/Smac was significantly increased(P<0.05).In cisplatin untreated group,the apoptosis rate was not associated with Smac expression.In cisplatin treated group(1,5,10 mg/L),compared with Eca109/neo and Eca109,the apoptosis rate of the Eca109/Smac was significantly increased after the treatments of cisplatin,the difference were significant(P<0.05).In addition,the apoptosis rate of Eca109/Smac was significantly increased with the concentration of cispaltin increased(P<0.05).The difference between Eca109/neo and Eca109 were not significant.CONCLUSION: Smac gene didn’t induce apoptosis in the cisplatin untreated Eca109 cells.Smac gene overexpression could increase chemotherapeutic sensitivity of esophageal cancer cell line Eca109 to cisplatin.