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

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(MTT)比色法检测GBC-SD、GBC-SD/EGFP和GBC-SD/Survivin 3种细胞的增殖活性;用逆转录-聚合酶链反应(RT-PCR)和Western blot测各组细胞中Survivin mRNA和蛋白质水平的表达;以合适浓度的顺铂(DDP,3.0 ms/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MIT法筛选IC_(50)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Western blot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IC_(50)[(2.03±0.24)ms/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Survivin shRNA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.

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

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 IC_(50) 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 IC_(50) 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 GBCSD/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.