南方医科大学学报

南方医科大学学报 ›› 2013, Vol. 33 ›› Issue (12): 1761-.

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细胞角蛋白8 GFP/Puro 双标干涉慢病毒系统的构建及对细胞凋亡的影响

金延超，尹荣华，郑巍薇，孔祥祯，詹轶群，杨晓明，李长燕   

  • 出版日期:2013-12-20 发布日期:2013-12-20

Construction of a GFP/Puro double-labeled lentiviral vector containing CK8 interfering
RNA and its effect on cell apoptosis in vitro

  • Online:2013-12-20 Published:2013-12-20

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摘要： ：目的构建细胞角蛋白8（CK8）的双标慢病毒干涉载体，获得高滴度慢病毒颗粒，感染HCT116细胞建立稳定株，检测CK8
敲低对细胞凋亡的影响。方法将CK8 的siRNA序列插入慢病毒表达载体GV248，并与包装质粒PMD、SPA共转染293T细
胞，36、48 h分两次收集慢病毒上清，应用流式细胞术检测病毒滴度。获得的病毒感染HCT116细胞，用嘌呤霉素筛选阳性细胞，
Western blotting检测干涉效果。采用Annexin V/PI染色检测干涉CK8对化疗药物顺铂诱导的细胞凋亡的影响。结果与结论
成功构建CK8干涉慢病毒载体并得到干涉稳定株，在HCT116细胞中干涉CK8 使细胞对顺铂引起的凋亡更加敏感。

Abstract: Objective To construct a GFP/Puro double-labeled lentiviral expression vector for CK8 silencing and assess the
effects of CK8 silencing on cell apoptosis. Methods The siRNA sequences of CK8 were inserted into the lentiviral expression
vector GV248 and transfected into 293T cells with the packaging plasmids PMD and SPA. The lentivirus was collected at 24
and 36 h post-transfection. Flow cytometry was used to detect the virus titer and the positive cells were selected with
puromycin. The knockdown of CK8 was examined by Western blotting. The effect of CK8 down-regulation on cell apoptosis
induced by cisplatin was detected with Annexin V/PI staining. Results and Conclusion We successfully constructed CK8
interference lentiviral vector and obtained a stable cell line with CK8 knock-down that was sensitive to cisplatin-induced
apoptosis.