论文部分内容阅读
目的构建肿瘤增殖基因Ki-67小干扰RNA(siRNA)表达质粒,研究其对人肾癌细胞Ki-67基因表达的阻抑作用,探索肾癌基因治疗新的途径。方法设计有小发夹机构的2条Ki-67siRNA对应模板DNA序列,煺火处理后克隆至pSliencer3.1质粒,构建重组质粒pSliencer-Ki-67。将pSliencer-Ki-67转染人肾癌786-0细胞,采用逆转录-聚合酶链反应(RT-PCR)、Westernblot检测Ki-67表达。结果酶切及测序证实质粒pSliencer-Ki-67构建成功。其可显著抑制786-0细胞Ki-67mRNA、Ki-67蛋白表达。结论成功构建的Ki-67siRNA表达质粒pSliencer-Ki-67能抑制人肾癌786-0细胞Ki-67基因表达。
Objective To construct a small interfering RNA (siRNA) expression plasmid for Ki-67 and to study its inhibitory effect on Ki-67 gene expression in human renal cell carcinoma, and to explore new ways of gene therapy for renal cell carcinoma. Methods Two template DNA sequences corresponding to Ki-67 siRNA were designed and cloned into pSliencer3.1 vector after heat treatment. The recombinant plasmid pSliencer-Ki-67 was constructed. PSliencer-Ki-67 was transfected into human renal carcinoma 786-0 cells. The expression of Ki-67 was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Results Digestion and sequencing confirmed that plasmid pSliencer-Ki-67 was successfully constructed. It can significantly inhibit Ki-67mRNA, Ki-67 protein expression in 786-0 cells. Conclusion The constructed Ki-67 siRNA expression plasmid pSliencer-Ki-67 can inhibit Ki-67 gene expression in human renal carcinoma 786-0 cells.