T7-shRNAs对SKOV3卵巢癌细胞株中C-erbB2基因的RNAi作用  

Apply specific silencing (RNAi)to interfere C-erbB2 expression in SKOV3 cells by T7-shRNAs

作　　者：朱宇[1] 程忠平[1] 戴虹[1] 

机构地区：[1]上海市杨浦区中心医院妇产科,200090

出　　处：《中国妇幼保健》2007年第18期2561-2564,共4页Maternal and Child Health Care of China

摘　　要：目的：探讨设计含有T7-启动子的Oligo-DNA，继而合成T7发夹RNA，并应用T7-shRNAs对卵巢癌SKOV3细胞株的卵巢癌C-erbB2基因进行RNAi。方法：自行设计并应用体外转录法合成、筛选出3条T7-shRNAs，脂质体转染卵巢癌SKOV3细胞株，分为非转染组、无关RNA组、shRNA（a、b、c）组，免疫细胞、RT-PCR及CCK-8观测干涉结果。结果：体外转录结果，合成后电泳检测shRNA（a、b、c）纯度较高，符合实验标准；转染后免疫细胞爬片结果，非转染组、无关RNA组和shRNAc链24、48、72h可见细胞生长正常，与时相无关，着色为深棕色，无抑制效果；shRNAa链24h可见有一定抑制效果，但48h即开始恢复生长，72h已恢复正常；shRNA b链24、48、72h可见有明显抑制效果，以24-48h最为明显，至72h仍维持相当抑制率；在核酸水平上半定量的RT-PCR结果与上述结果相符合；CCK-8动态观测，经shRNA干涉后不同时间内的抑制曲线显示，shRNAb链的抑制作用最明显，而shRNAa链和shRNAc链的抑制作用不明显（P〈0.01），但shRNAa链抑制作用强于shRNAc链（P〈0.01），从整个时间段来看，shRNAb在48h的抑制作用最强。结论：研究证实应用发夹RNA对肿瘤细胞可以取得明显干涉效果，发现shRNAb对C-erbB2基因干涉具有明显抑制作用，进一步完善RNA干涉实验来抑制肿瘤细胞生长是存在可能的。Objective： To study how to design T7 - promotor - Oligo - DNAs and compose short - hairpin RNA. Then evaluate the effect of RNA interference that used T7 - ShRNAs transfer SKOV3 cells. Methods： we designed the oligo - DNA template which contains T7 - promotor according to C - erbB2 exons. By groping the annealing temperature , we designed the hairpin sequence. Then we used it to evaluate the inhibitory affection of shRNA, after we transected it into SK - OV3 cells. To apply CCK - 8 to count the cell growth dynamic so that observe the inhibitory affection. We could use immuno -histo -chemistry, semi -quantization PCR and Western -Blot to evaluate the inhibitory affection. Results： RNAi resulted by RT -PCR, immuno -histo -chemistry cells print and CCK -8 ： inhibiting rate shRNAb 〉 shRNAa （ P 〈 0. 01 ）, it was no effect in shRNAc, no - transfect group and irrespective - RNA group; after 24 h shRNAa＇s inhibiting was resumed, it was get right at 72 h. we found that shRNAb＇s inhibiting affection was most remarkable , especially in 48h; even at 72 h, its inhibitory affection was retained. Conclusion： shRNAb＇s inhibiting is successfully, and it is possible to use siRNA to effetely interfere the expression of C - erbB2 in tumor cells so that settle the base that we can cure the ovarian cancer in the future.

关 键 词：卵巢癌 C -erbB2 SKOV3 RNA干涉 

分 类 号：R737.31[医药卫生—肿瘤]