【摘要】 目的研究针对Ki67基因的短发夹状RNA(shRNA)表达载体pSilencer-Ki67对人肾癌细胞生长的抑制作用。方法将pSilencer-Ki67转染人肾癌786-0细胞,24、48、72、120、144 h后分别采用逆转录-聚合酶链反应(RT-PCR)、Western blot技术检测Ki67 mRNA、蛋白表达,噻唑蓝(MTT)比色法检测细胞增殖,原位末端标记法检测细胞凋亡。结果pSilencer-Ki67转染后48 h 786-0细胞Ki67mRNA表达(34．1±1．7)%、蛋白表达(42．6±1．7)%降低最明显,与空质粒对照组[(97．7±0．9)%、(97．3±1．6)%]比较差异均有统计学意义(P＜0．01)。pSilencer-Ki67处理后48 h细胞增殖抑制率(76．7±3．2)%最高,72 h凋亡细胞阳性率(74．3±5．9)%最高,与空质粒对照组[(7．0±0．5)%、(8．4±1．1)%]比较差异有统计学意义(P＜0．01)。pSilencer-Ki67的这些作用可持续144 h。结论pSilencer-Ki67能有效、持续抑制人肾癌786-0细胞生长,有望成为肾癌基因治疗的有效工具。更多还原

【Abstract】 Objective To evaluate inhibitory effects of the recombinant plasmid of small hairpin RNA(shRNA)against Ki67 gene,named pSileneer-Ki67,on the growth of human renal carcinoma 786- 0 cells.Methods The pSilencer-Ki67 was transfected into human renal carcinoma cell line 786-0 cells.At 24,48,72,120 and 144 h posttransfection,the effects of pSilencer-Ki67 on the Ki67 expression of human renal carcinoma 786-0 cells were detected by RT-PCR and Western blot.The proliferation of 786-0 cells was measured by MTT assay.The apoptosis of 786-0 cells was tested by TUNEL assay.Results RT- PCR and Western blot analyses revealed that the Ki67 expression levels of 786-0 cells treated with pSi- lencer-Ki67 were reduced significantly.The proliferation of 786-0 cells treated with pSilencer-Ki67 was inhibited and the apoptosis increased.The knock down of Ki67 and inhibition of proliferation and indue- tion of apoptosis mediated by pSileneer-Ki67 were maintained for 144 h and the maximal effect achieved at 48 h or 72 h posttransfection.Conclusion The expression plasmid of shRNA against Ki67 gene can inhibit the proliferation and induce apoptosis for a long period by blocking Ki67 expression of human renal eareinoma cells.The inhibition of Ki67 expression by pSileneer-Ki67 may be a rational approach in gene therapy for renal cancer.更多还原