【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。更多还原

【Abstract】 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.更多还原