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目的探讨HPV16E6小干扰RNA(siRNA)与宫颈癌CaSki细胞中E6、p53、p21之间的关系。方法2004年9月至2005年3月于四川大学华西第二医院,应用化学合成针对HPV16E6的siRNA借脂质体转染CaSki细胞,实时荧光定量逆转录-聚合酶链反应(RT-PCR)和流式细胞术检测E6siRNA转染前后细胞中HPV16E6、p53、p21mRNA及其蛋白表达的变化。结果转染24h,E6mRNA的表达显著低于空白组(P<0.05)。各时间点p53、p21mRNA的表达差异无显著性意义(P>0.05)。转染48h,E6蛋白表达明显下调,p53、p21蛋白表达相应升高。结论HPV16E6siRNA能特异、高效地沉默宫颈癌细胞E6mRNA的表达,减少对野生型p53的降解,恢复p53蛋白的功能活性。RNA干扰(RNAi)技术可为HPV感染相关性疾病提供一种新的特异性基因治疗方法。
Objective To investigate the relationship between HPV16E6 small interfering RNA (siRNA) and E6, p53, p21 in cervical cancer CaSki cells. Methods From September 2004 to March 2005, siRNAs targeting HPV16E6 were synthesized and transfected into CaSki cells by lipofectamine. Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Flow cytometry was used to detect the expression of HPV16E6, p53, p21 mRNA and protein in E6 siRNA before and after transfection. Results After transfection for 24 hours, the expression of E6 mRNA was significantly lower than that of the blank group (P <0.05). There was no significant difference in the expression of p53 and p21mRNA between different time points (P> 0.05). 48h after transfection, E6 protein expression was significantly down-regulated, p53, p21 protein expression increased accordingly. Conclusion HPV16E6 siRNA can specifically and efficiently silence the expression of E6 mRNA in cervical cancer cells, reduce the degradation of wild type p53 and restore the functional activity of p53 protein. RNA interference (RNAi) technology can provide a new and specific gene therapy for HPV-related diseases.