CAJ | 학술논문

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。
목적：구건세포각단백8（CK8）적쌍표만병독간섭재체，획득고적도만병독과립，감염HCT116세포건립은정주，검측CK8고저대세포조망적영향。방법장CK8적siRNA서렬삽입만병독표체재체GV248，병여포장질립PMD、SPA공전염293T세포，36、48 h분량차수집만병독상청，응용류식세포술검측병독적도。획득적병독감염HCT116세포，용표령매소사선양성세포， Western blotting검측간섭효과。채용Annexin V/PI염색검측간섭CK8대화료약물순박유도적세포조망적영향。결과여결론성공구건CK8간섭만병독재체병득도간섭은정주，재HCT116세포중간섭CK8사세포대순박인기적조망경가민감。
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.