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目的:探讨运用慢病毒载体介导的RNA干扰技术对X-连锁凋亡抑制蛋白(XIAP)的抑制效率及对胰腺癌细胞增殖、凋亡的影响,建立XIAP表达稳定抑制的胰腺癌细胞株。方法:应用pGCSIL-PUR慢病毒载体构建针对XIAP的ShRNA载体,转染包装细胞293T,收集病毒上清转染胰腺癌细胞系SW1990,经嘌呤霉素(puromycin)筛选并扩大培养得到稳定克隆;实时荧光定量PCR和western-blot免疫印迹检测癌细胞内XIAP的表达;四甲基偶氮唑盐(MTT)比色法检测细胞增殖;caspase 3/7活性测定和DAPI染色检测细胞凋亡。结果:成功构建3个XIAP-ShRNA慢病毒载体(X1、X2、X3)及XIAP表达稳定抑制的胰腺癌细胞株,对XIAP的抑制效率均达70%以上;MTT检测显示X1、X3稳定抑制XIAP后胰腺癌细胞增殖明显减慢,但caspase 3/7活性及细胞凋亡并没有明显增加。结论:慢病毒载体介导的靶向XIAP的RNAi可有效抑制XIAP表达,降低胰腺癌细胞的增殖能力;成功建立的XIAP表达稳定抑制的胰腺癌细胞株为进一步研究打下基础。
OBJECTIVE: To investigate the inhibitory effect of lentiviral vector-mediated RNA interference on X-linked inhibitor of apoptosis (XIAP) and its effect on the proliferation and apoptosis of pancreatic cancer cells, and to establish a pancreatic cancer cell line with stable expression of XIAP. METHODS: ShRNA vector targeting XIAP was constructed by pGCSIL-PUR lentiviral vector, transfected into 293T cells, and then transfected into 293T cells. The supernatants were transfected into SW1990 cells and screened by puromycin to obtain stable clones. The expression of XIAP in cancer cells was detected by real-time quantitative PCR and Western-blot. The cell proliferation was detected by MTT colorimetric assay. The apoptosis was detected by caspase 3/7 activity assay and DAPI staining. RESULTS: Three XIAP-ShRNA lentiviral vectors (X1, X2, X3) and pancreatic cancer cell lines with stable expression of XIAP were successfully constructed, and their inhibitory efficiency against XIAP was over 70%. MTT assay showed that XIAP and X3 could inhibit XIAP Proliferation of pancreatic cancer cells significantly slowed down, but caspase 3/7 activity and apoptosis did not significantly increase. CONCLUSION: Lentiviral vector-mediated RNAi targeting XIAP can effectively inhibit the expression of XIAP and reduce the proliferation of pancreatic cancer cells. The established pancreatic cancer cell lines with stable XIAP expression lay the foundation for further study.