pEGFP—Survivin对GBC—SD细胞生长的抑制及对化疗敏感性的影响

The effect of Survivin shRNA on chemotherapy resistance of GBC-SD cells

目的观察重组质粒pEGFP—Survivin对胆囊癌细胞（GBC—SD）化疗敏感性的影响。方法以噻唑蓝（MTr）比色法检测GBC-SD、GBC-SD／EGFP和GBC—SD／Survivin3种细胞的增殖活性；用逆转录-聚合酶链反应（RT—PCR）和Westernblot测各组细胞中SurvivinmRNA和蛋白质水平的表达；以合适浓度的顺铂（DDP，3．0mg／L）作用相同的时间后，用MTY法检测3种细胞存活率，流式细胞仪测细胞凋亡，并用MTT法筛选IC50值，TUNEL法观察细胞核改变；另外检测DDP作用后各组细胞的Caspase．3蛋白酶活性的变化。结果GBC—SD和GBC—SD／EGFP细胞的增殖活性大致相同，而GBC—SD／Survivin细胞的增殖活性明显下降；RT—PCR和Westemblot均发现GBC—SD／Survivin细胞中的Survivin表达水平较其余两种细胞明显下降（分别下降了74．7％和71．5％）；在给予了DDP作用后，GBC—SD／Survivin细胞存活率和IC50[（2．03±0．24）mg／L]明显较低，细胞凋亡率较高（84．3％），而3种细胞用TUNEL法染色后均可见棕色凋亡细胞核。给予了DDP作用后，各组细胞Caspase-3活性均呈现先升高后下降的趋势，但GBC—SD／Survivin细胞中Caspase-3的活性明显高于另外两种细胞。结论pEGFP—Survivin表达的SurvivinshRNA能明显降低GBC—SD细胞中Survivin的表达．提高对化疗药物的敏感性。

Objective To investigate the effect of Survivin shRNA on chemotherapy resistance of GBC-SD cells. Methods They were divided into three group which are GBC-SD, GBC-SD/EGFP and GBC-SD/survivin. MTT assay was used to detect cells viability in three groups, and mRNA and protein of survivin were tested by RT-PCR and Western blot. Then cells were treated with proper construction DDP （3.0 mg/L） for the same time,cell survival rate and IC50 was detected with MTT respectively,cells apoptosis was detected through FACS, and the alteration of nucleus was observed by TUNEL. In addition, caspase-3 activity was detected by using colorimetric method. Results Cell viability was reduced remarkably in GBC-SD/survivin and survivin expression was decreased obviously （74.7% ,71.5% ）. After treated with DDP,cell survival rate and IC50 was decreased obviously [ （2.03 ±0.24） mg/L] in GBC-SD/ survivin, apoptosis rate （84.3%） were elevated remarkably compared with other group. There were brownly nucleuses in three groups. Caspase-3 activity all ascend first and then descent,but it exceeded in GBC- SD/survivin than that in other two groups. Conclusion The survivin shRNA could down-regulate the expression of survivin in GBC-SD cells remarkably and improve the sensibility to chemotherapy. The alteration of caspase-3 activity followed with survivin contrarily.