siRNA抑制survivin基因周期的影响表达对HeLa细胞

Influence of survivin gene expression inhibition with siRNA on the cell cycle of HeLa cell line

目的设计靶向survivin基因的干涉片段，构建真核表达载体pSUPER／survivin并转染宫颈癌HeLa细胞，探讨siRNA抑制survivin表达对联合1射线宫颈癌HeLa细胞周期的影响。方法化学合成干涉引物，退火获得survivin基因的干涉片段，连入pSUPER载体并测序。将测序正确的pSUPER／survivin载体转染HeLa细胞株，RT—PCR法和流式细胞术检测HeLa细胞中survivin基因的表达，流式细胞术检测转染联合＇射线照射后各组细胞的细胞周期数据。结果本实验成功的构建了pSUPER／survivin载体。转染pSUPER／survivin的HeLa细胞其survivin基因表达水平明显低于未转染的HeLa细胞及转染空载体pSUPER的HeLa细胞。1射线照射后，转染pSUPER／survivin的HeLa细胞发生了S期细胞比例下降，及G2／M期阻滞。结论成功的构建了pSUPER／survivin载体，siRNA干涉survivin可以作为提高放射敏感性的潜在分子靶点，通过减少S期细胞比例，促进肿瘤细胞凋亡，通过增加G2／M期阻滞增加放射线的杀伤。

Objective To design siRNA targeting to survivin and construct an eukaryoeytic expression vector pSUPER/survivin, then transfected it into cervical cancer cell line HeLa and evaluated the effects of siRNA against survivin gene on cell cycle of cervical cancer cell line HeLa irradiated with γ - rays. Methods The primer was synthesized, siRNA targeting to survivin was obtained,linked with the vector pSUPER and sequenced. The correet pSUPER/survivin was transfected into HeLa cells and the expression of survivin gene in Hela cells was detected by RT- PCR and Flow Cytometry（FCM）. In the present study, the effects of survivin gene transfeetion combined with irradiation on cell cycle distribution were analysed by FCM. Result Recombinant eukaryocytic expression vector pSUPER/survivin was successfully constructed. FCM results indicated that the expression of sur vivin gene is significantly suppressed in pSUPER/survivin transfected cells compared with non -transfected cells or empty vector pSUPER transfected cells. After γ-ray irradiation, HeLa cells transfeeted with pSUPER/survivin induced less proportion of S phase and G2/M arrest. Conclusion Recombinant eukaryoeytie expression vector pSUPER/survivin was successfully constructed, siRNA against survivin enhanced radiosensitivity through execution of apoptosis,induction less proportion of S phase and G2/M arrest,it may be a potential target for improving radiosensitivity