靶向抑制survivin对胰腺癌细胞增殖和凋亡效应的影响

目的 探讨反义survivin—脂质体复合物对胰腺癌细胞增殖和凋亡效应的影响及机制,为胰腺癌及survivin阳性肿瘤的治疗提供理论依据。方法 用脂质体介导survivin反义寡核苷酸转染胰腺癌细胞系PANC—1细胞;通过RT—PCR、Western印迹试验检测survivin表达水平;MTY法测量细胞生长情况;流式细胞术测定caspase—3活性及凋亡率;电镜观察细胞形态学变化;应用激光共聚焦显微镜免疫荧光分析法评价脂质体反义复合物在亚细胞水平对survivin蛋白分子的影响。结果 反义survivin脂质体复合物有效下调survivin表达水平,并呈剂量依赖关系,IC50值为300nmoL／L,最大效应浓度为500nmoL／L,此时表达水平下调了80％。类似细胞生长抑制结果为MTY实验所证实。与此同时,caspase—3活性和凋亡率随转染时间延长而逐渐递增,并出现凋亡形态学变化,如胞浆空泡变性、核浓缩和核碎裂。免疫荧光分析,标记有FITC—绿荧光染色的survivin蛋白分子在未转染的细胞浆中清晰可见,并呈“斑点”状分布;而在转染细胞中几乎没有发现,但是符合细胞凋亡的形态学特征。结论 靶向抑制联系细胞增殖和凋亡的关键分子—survivin在胰腺癌治疗中有良好的应用前景。

Effect of survivin targeting on cell proliferation and apoptosis in pancreatic cancer cells

Objective To investigate the effects of antisense survivin-Lip compound on pancreatic cancer cell proliferation and apoptosis and its mechanism. Methods Pancreatic cancer cells of the line PANC-1 were cultured. Survivin oligonucleotide (ODN) was transfected into the PANC-1 cells mediated by Lip reagent. The expression of survivin mRNA and that of surviving protein were detected by RT-PCR and Western blotting. MTT assay was applied to determine the proliferation of PANC-1 cells. Active caspase-3 and apoptosis rate were evaluated by flow cytometric analysis. The morphological changes were assessed by electron microscope. Lascar scanning confocal microscope immunofluorescence analysis was performed to detect the subcellular localization of survivin protein on treated cells and untreated cells. Results Antisense compound efficiently down-regulated the survivin expression (mRNA and protein) in dose-dependent manner with an IC50 of 300 nmol/L. Its maximum effect was achieved at the concentration of 500 nM, at which the expression level was down-regulated by 80%. The similar results were found in MTT assay. As revealed by gradually increased caspase-3-like protease activity and apoptosis rate in a time-dependent manner, and the morphological changes of apoptosis such as blebbing and loss of microvilli, vacualization in cytoplasm, condensation of cytoplasm and nucleus, and fragmented chromatin, treatment with antisense compound induced apoptosis and inhibited cell growth. Fluororescein isothiocyanate (FITC)-labeled immunofluorescence staining of survivin clearly showed that survivin was expressed mainly in the formation of a spotted distribution inside the cytoplasm of untreated cells. Survivin protein molecules were clearly seen in the cytoplasm of the untransfected cells and distributed like spots and almost disappeared in the transfected cells with morphological changes conforming to the changes of apoptosis. Conclusion Survivin protein is a key molecular connecting proliferation with apoptosis and antisense oligonucleotides targeting survivin have a bright prospect in therapy of pancreatic cancer cells.