摘要
目的研究核因子(NF)-кB诱骗(decoy)寡核苷酸对肝癌耐药细胞株HepG2／阿霉素(ADM)化疗敏感性的影响及其内在机制。方法培养肝癌耐药细胞株,转染FITC标记的NF-кB decoy寡核苷酸,通过荧光显微镜和共聚焦显微镜进行核内定位的观察,凝胶迁移率实验检测转染前后NF-кB结合活性的变化,加入ADM,噻唑蓝(MTT)比色法检测细胞增殖,采用流式细胞仪和TUNEL法检测细胞凋亡,观察转染NF-кB decoy寡核苷酸后肝癌耐药细胞对化疗药物的敏感性变化,DEVD-pNA降解法检测Caspase-3的活性变化,Western blot检测bcl-2蛋白表达的变化。结果转染荧光标记的NF-кB decoy寡核苷酸1 h,倒置荧光显微镜和共聚焦显微镜显示定位于细胞核内,凝胶阻滞分析实验(EMSA)分析示NF-кB decoy寡核苷酸能够降低NF-кB核内结合,加入化疗ADM(0．1 mg/L)后,ADM作用24 h后出现细胞凋亡,流式细胞术、TUNEL法检测NF-кB decoy寡核苷酸转染HepG2／ADM细胞的凋亡,与对照组相比,凋亡指数增加,Caspase-3的活性增加,bcl-2的表达下调。结论NF-кB decoy寡核苷酸转染肝癌耐药细胞株HepG2／ADM后,能够抑制NF-кB的激活,Caspase-3活性增加和bcl-2的表达下调是其增加化疗药物的敏感性的可能机制。
Objective To investigate a double-stranded decoy oligodeoxynueleotides （ODNs） with a speeifie affinity to NF-κB effeetively suppressed NF-κB aetivity and sensitized hepatie eaneer eells to ehemotherapy. Methods HepG2 eells resistanting to adriamyein （HepG2/ADR） were indueed stepwised,FITC-labeled decoy ODNs against NF-κB were transfeeted into HepG2/ADR eells with LipofectAMINE^TM2000, Inverse fluoreseent mieroseopand showed the localization of decoy DNA, and EMSA was performated to investigate the affinity of NF-κB. ADM （0.1 mg/L） was added, the apoptosis was observed by flow eytometry and TUNEL differently. The aetivity of easpase3 was found by quantitative assay,the expression of bel-2 protein was deteeted by western blot. Results HepG2/ADR was confirmed resisting to ADM. When decoy ODNs was transfeeted, after 1 hour, FITC-labeled decoy ODNs against NF-κB was deteeted in the nuclei of HepG2/ADM eells, and eleetrophoretie mobility shift assays （EMSA） was performed to found the aetivation of NF-κB binding to the nueleus was inhibitd. Ineubed with ADM （0.1 mg/L） ,it showed signifieant inhibitory effeet on the growth of HepG2/ADM,the pereentage of apoptosis was increased compare with control at 24 h,easpase3 activity was inereased,bel-2 expression was downregulated. Conclusion Our findings suggest that decoy ODNs for NF-κB could be a novel and attractive strategy to improve treatment outcome of human hepatocarcinoma,bcl-2 and caspase3 could play important role during decoy ODNs inducing apoptosis.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2006年第9期1075-1078,共4页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金(30400431)
