Smac蛋白干扰胰腺癌Bx-PC3细胞系survivin蛋白增敏顺铂化疗

Interference of survivin in pancreatic cancer Bx-PC3 cell line with Smac sensitizing its response to cisplatin

目的:观察Smac蛋白对细胞凋亡抑制蛋白survivin的下调作用,进一步分析Smac蛋白转染增敏survivin过表达胰腺癌Bx-PC3细胞系顺铂化疗敏感性的效应。方法:建立稳定传代、均一表达survivin的Bx-PC3细胞系。制备Smac-GFP重组质粒,将其转染入Bx-PC3细胞,以Western blot法半定量测定survivin表达水平。联合10mg/L顺铂孵育48h和72h,按照MTT法测定细胞抑制率(%)。结果:Bx-PC3细胞转染24h开始出现微弱阳性转染表现(GFP绿色荧光阳性),于48h出现大量细胞转染,于72h转染水平达到峰值。Western blot检测表明,与对照组相比,转染组的survivin蛋白表达水平仅为对照组的28.6%(P<0.01)。MTT检测发现,随着顺铂作用时间的延长,对照组和转染组的顺铂抑制率均升高(48hvs72h,P<0.05)。而且,转染组的抑制率在两时点上均高于对照组(P<0.01)。结论:Survivin蛋白在胰腺癌中具有重要的增殖调控作用,并且与胰腺癌化疗抵抗密切相关。Smac蛋白拮抗可以在蛋白水平上下调survivin的表达,提高胰腺癌细胞对化疗的敏感性,这一治疗策略有望成为一种治疗胰腺癌的新型有效治疗方法。

AIM：To investigate the downregulation of survivin,an inhibitor of apoptosis protein with Smac and further analyze the sensitization to cisplatin chemotherapy in survivin-overexpressing pancreatic cancer BX-PC3 cell line with Smac transfection. METHODS：Stably passaged Bx-PC3 cell line was established to express survivin uniformly. Smac-GFP recombinant plasmid was constructed and transfected into BX-PC3 cells,in which survivin was semiquantitatively analyzed by Western blot. Cells were incubated with 10 mg/L cisplatin for 48 h and 72 h,whose rate of inhibition （%） was determined with MTT assay. RESULTS：Bx-PC3 cells presented mild positive transfection （green GFP fluorescence positive） at 24 h post transfection,which became substantial at 48 h and reach its peak at 72 h. Western blot assay showed that survivin in transfected cells was equal to 28.6% of that in control cells （P〈0.01）. Moreover,with the prolongation of cisplatin treatment,both control and transfected cells showed a rising trend in their rates of inhibition （48 h vs 72 h,P〈0.05） and meanwhile the former was always below the latter at both time points （P〈0.01） as confirmed by MTT assay. CONCLUSION：Survivin played an important role in regulation of proliferation in pancreatic cancer and is also closely correlated to its chemotherapeutic resistance. Antagonism with Smac could downregulate the protein expression of survivin and sensitize the response of pancreatic cancer to chemotherapeutic agents,which emerged as a potential and effective therapeutic strategy for pancreatic cancer.