摘要
目的研究特异小干扰RNA(sm all interfering RNA,siRNA)对宫颈癌HeLa细胞中人乳头瘤病毒(hum an pap-illom avirus,HPV)18型E6基因的抑制及其对细胞凋亡的影响。方法针对HPV18E6基因设计siRNA序列,经PCR方法体外扩增,得到含有U6启动子以及siRNA序列的PCR产物,利用L ipofectam ineTM2000脂质体转染HeLa细胞,在U6启动子的作用下于细胞内转录siRNA。针对转染后不同时间点采用四唑盐(MTT)比色法测定细胞活力,流式细胞仪PI染色法检测细胞凋亡率,RT-PCR测定HPV18E6 mRNA变化。结果转染siRNA后细胞活力受到显著抑制(P<0.05),光镜下出现明显的凋亡形态,72 h的凋亡率达到55.8%。RT-PCR结果显示,细胞转染24、48和72 h后HPV18E6 mRNA分别减少了57%、78%和40%,而siRNA阴性对照与未转染细胞相比差异不显著。结论siRNA可特异有效的干扰宫颈癌HeLa细胞内HPV18E6基因的表达,从而可诱导肿瘤细胞凋亡。
Objective To study the effect of small interfering RNA （siRNA） of human papillomavirus （HPV） 18 E6 gene on apoptosis of HPV-related cervical HeLa cell line. Methods siRNA targeting HPVI8 E6 mRNA was designed and generated by PCR amplification. The PCR products containing U6 promoter and the siRNA sequence were then transfected into HeLa cells via Lipofectamine m2000. Cell viability was determined by MTr assay. Apoptosis was detected by morphological observation and flow cytometry analysis. The expression level of HPV18 E6 mRNA was assayed by RT-PCR. Results The cell growth and viability of siRNA transfected group were significandy inhibited（P 〈 0. 05 ）. There were obvious morphological changes in HeLa cells after transfection of siRNA as shown by microscopy and the apoptosis rate at 72 h was 55.8%. HPVI8 E6 mRNA expressions in the cells of the interference group at 24 -48 and 72 h after transfection were significantly reduced by about 57% -78% and 40% respoctively, as compared with that of the negative and blank control groups, whereas the latter two groups had similar expression levels. Condusions Interference with siRNA against HPV18 E6 gene can effectively inhibit the specific gene expression, which may play a significant role in the induction of apoptosis of the corresponding HeLa cells.
出处
《基础医学与临床》
CSCD
北大核心
2006年第10期1112-1117,共6页
Basic and Clinical Medicine
基金
广州市重大科技攻关项目(01-Z-005-01)
