Smac基因过表达对宫颈癌HeLa细胞放疗敏感性的影响

Effects of Smac gene over-expression on radiotherapeutic sensitivity of cervical cancer cell line HeLa

目的探讨转染Smac基因过表达对宫颈癌细胞放疗敏感性的影响,寻求改善宫颈癌放疗疗效的新途径。方法将Smac基因转染入宫颈癌HeLa细胞株,G418筛选获得亚克隆细胞,RTPCR、Westernblot法检测癌细胞Smac基因表达。X射线照射后,采用MTT比色法检测癌细胞体外生长活性;透射电镜、AnnexinV-FITC和碘化丙啶双染色流式细胞仪法检测癌细胞凋亡及比率;Westernblot、比色法检测细胞内Caspase-3蛋白表达和活性水平。结果RTPCR和Westernblot证实所获宫颈癌亚克隆细胞HeLaSmac的SmacmRNA、蛋白表达水平均显著高于HeLa(P<0.01)。8GyX射线照射后,HeLaSmac细胞生长活性较HeLa细胞减弱10.19%(P<0.01),透射电镜下可见部分HeLaSmac细胞发生典型凋亡形态学改变,凋亡率为16.4%,显著高于对照组HeLa细胞(凋亡率为6.2%,P<0.01)。与HeLa细胞比较,HeLaSmac细胞内Caspase3表达显著增加(P<0.01),活性水平提高3.42倍(P<0.01)。结论稳定转染外源性Smac基因使其在宫颈癌细胞株中过表达,能提高癌细胞中Caspase3的表达和活性,增强放疗对癌细胞的诱导凋亡作用,有望成为改善宫颈癌放疗效果的新途径。

Objective To study the effects of extrinsic Smac gene transfection and its over-expression on radiotherapeutic sensitivity of cervical cancer cells,in order to explore a novel strategy for ameliorating radiotherapy of cervical cancer. Methods After Smac gene was transferred into cells of cervical cancer cell line HeLa,the subclone cells were obtained by persistent G_(418) selection.Cellular Smac gene expression was determinated by RT-PCR and Western blot.After treatment with X-ray irradiation,cellular growth activity in vitro was investigated by MTT colorimetry.Cellular apoptosis and its rate were determinated by electronmicroscopy,Annexin V-FITC and propidium iodide staining flow cytometry.Cellular Caspase-3 protein expression and its activity were assayed by Western blot and colorimetry. Results RT-PCR and Western blot demonstrated that Smac mRNA and protein levels of HeLa/Smac cells,the selected subclone cells of cervical cancer cell line,were significantly higher than those of HeLa cells (P<0.01).After treated with 8 Gy X-ray irradiation,growth acitivity of HeLa/Smac cells reduced by 10.19%(P<0.01),as compared with that of HeLa cells.Partial HeLa/Smac cancer cells presented characteristic morphological changes of apoptosis under electronmicroscope,with an apoptosis rate of 16.4%,which was significantly higher than that of HeLa cells(6.2%,P<0.01).Compared with HeLa cells,Caspase-3 expression level in HeLa/Smac was improved significantly(P<0.01),while its activity was 3.42 times as much as that of HeLa cells(P<0.01). Conclusion Stable transfection of extrinsic Smac gene and its over-expression in cervical cancer cell line could significantly enhance cellular caspase-3 expression and activity,ameliorate apoptosis-inducing effects of radiation on cancer cells,which would be a novel strategy to improve radiotherapeutic effects for cervical cancer.;