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目的:应用慢病毒介导的RNA干扰技术,建立Clusterin(CLU)基因稳定干扰的人肾癌细胞系,为CLU基因的功能研究奠定基础。方法:以人CLU mRNA编码序列作为干扰靶点,与pGCSIL-GFP载体连接真核表达载体。另外构建不针对任何已知mRNA的阴性对照shRNA表达质粒。利用包装细胞293T获得重组慢病毒,分别感染人肾癌786-O细胞株及ACHN细胞株。应用Real time-PCR及Western blot检测不同组别CLU表达的变化。结果:成功构建CLU shRNA慢病毒载体CLU-RNAi-LV并获得相应的慢病毒,病毒悬液滴度>4×1011TU/L。RealTime-PCR、Western blot结果显示干扰组CLU mRNA及蛋白表达水平较对照组显著降低。结论:慢病毒介导的CLU基因干扰能稳定有效地表达于肾癌细胞,筛选出能稳定干扰CLU基因表达的siRNA序列和肾癌786-O细胞株。
OBJECTIVE: To establish a human renal cell carcinoma cell line with stable interference of Clusterin (CLU) gene by lentivirus-mediated RNA interference technology, and lay a foundation for the study of CLU gene function. Methods: Human CLU mRNA coding sequence was used as the interference target, and eukaryotic expression vector was ligated with pGCSIL-GFP vector. In addition, negative control shRNA expression plasmids that do not target any known mRNAs were constructed. Recombinant lentivirus was obtained by packaging 293T cells and infected human renal carcinoma cell line 786-O and ACHN respectively. The changes of CLU expression in different groups were detected by Real time-PCR and Western blot. Results: The CLU shRNA lentiviral vector CLU-RNAi-LV was successfully constructed and the corresponding lentivirus was obtained. The titer of virus suspension was> 4 × 1011 TU / L. RealTime-PCR and Western blot showed that CLU mRNA and protein expression in the interference group were significantly lower than those in the control group. CONCLUSION: Lentivirus-mediated CLU gene knockdown can be stably and efficiently expressed in renal cancer cells. SiRNA sequences stably interfering with CLU gene expression and renal cancer 786-O cell lines were screened out.