RNA干涉与卵巢癌化疗药物敏感性的相关性研究

Correlative study to specific silencing (RNAi) by T7-shRNAs and sensitivity of chemotherapy in ovarian cancer

目的设计应用含有T7-启动子的Oligo-DNA合成发夹RNA,并应用T7-ShRNAs对卵巢癌SKOV3细胞株的C-erbB2基因进行RNAi,继而进行与卡铂相关的化疗敏感性研究。方法自行设计并应用体外转录法合成并筛选出3条T7-ShRNAs,脂质体转染卵巢癌SKOV3细胞,分为非转染组、无关RNA组、shRNA(a,b,c)组,免疫细胞、RT-PCR观测干涉结果,应用CCK-8进行干涉与化疗敏感性的监测。结果体外转录。结果合成后电泳检测shRNA(a,b,c)纯度符合实验标准;转染后免疫细胞爬片结果及半定量的RT-PCR结果显示:非转染组、无关RNA组和shRNAc链24、48、72小时可见细胞生长正常,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48小时即开始恢复生长,72小时已恢复正常;shRNAb链24、48、72h可见有明显抑制效果,以24-48小时最为明显,至72小时仍维持相当抑制率;CCK-8动态观测:经shRNA干涉后不同时间内的抑制曲线显示:shRNAb链的抑制作用明显,与卡铂联合应用效果明显抑制作用最强。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,通过RNA干涉关闭基因后可明显增加卡铂敏感性。其机制尚需进一步探讨。

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 and correlative study with sensitivity of chemotherapy（CBP）.Methods Designed the oligo-DNA template which comains T7-promotor according to C-erbB2 exons,designed the hairpin sequence. Then used it to evaluate the inhibitory effection of shRNA, after we transfect it into SK-OV3 cells. Used immano-histo-ehemistry, semi-quan- titation PCR to evaluate the the inhibitory effection, then Apply CCK-8 to count the cell growth dynamic so that observe the inhibitory effection and sensitivity of chemotherapy（CBP）.Results RNAi result by RT-PCR, immuno-histo-ehemistry cells prim and CCK-8： inhibiting rate shRNAb〉 shRNAa（ P 〈 0.01）,it＇s no effect in shRNAe,no-transfeet group and irrespective-RNA group;after 24 h shRNAa＇ s inhibiting was resumed, it was get right at 72 h, that shRNAb＇ s inhibiting effection is remarkable, especially in 48 h; even at 72 h,its inhibitory effection was retained,when RNAi associated with CBP the inhibitory effection was most remarkable. Condusion shRNAb＇ s inhibiting is successfully, and it is possible to use siRNA to effectly interfere the expression of C-erbB2 in tumor cells so that settle the base that RNAi can made gene dysfimetion,and ameliorate the sensitivity of ehemotherapy（CBP）.