NF-κBdecoy寡核苷酸增强阿霉素对肝癌耐药细胞株HepG_2/ADM化疗敏感性的研究  

A double strand decoy DNA oligonucleotides for NF-κB induced apoptosis and sensitization of hepatic cancer cells to chemotherapy

作　　者：严斌[1] 陈孝平[1] 张万广[1] 兰明银[2] 

机构地区：[1]华中科技大学同济医学院附属同济医院肝脏外科中心,武汉市430030 [2]湖北省十堰市太和医院肝胆移植外科,十堰市442000

出　　处：《中华肝胆外科杂志》2004年第10期691-695,共5页Chinese Journal of Hepatobiliary Surgery

基　　金：卫生部临床重点学科项目 (2 0 0 1)

摘　　要：目的　研究NF κBdecoy寡核苷酸增强肝癌耐药细胞株HepG2 /ADM对阿霉素化疗敏感性的变化。方法　通过培养液中阿霉素 (adriamycin ,ADM)的浓度梯度增加法 ,长期筛选培养 ,建立肝癌HepG2 /ADM耐药细胞株 ,荧光定量PCR检测MDR1的表达 ,转染FITC标记的NF κBdecoy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 κB结合活性的变化 ,加入化疗药物阿霉素 (ADM) ,MTT比色法检测细胞增殖 ,细胞凋亡采用流式细胞仪和TUNEL法检测观察转染NF κBdecoy寡核苷酸后肝癌耐药细胞对化疗药物的敏感性变化。结果　HepG2 /ADM细胞是一个明确的多药耐药细胞模型 ,转染荧光标记的NF κBdecoy寡核苷酸 1h ,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 (electrophorereticmobilityshiftassay ,EMSA)分析示NF κBdecoy寡核苷酸能够降低NF κB核内结合 ,加入化疗ADM(0 1mg/L)后 ,MTT显示HepG2 /ADM细胞的增殖明显抑制 ,ADM作用 2 4h后出现细胞凋亡 ,流式细胞术、TUNEL法检测NF κBdecoy寡核苷酸转染HepG2 /ADM细胞诱导的凋亡 ,与对照组相比 ,凋亡指数增加。结论　NF κBdecoy寡核苷酸转染肝癌耐药细胞株HepG2 /ADM后 ,能够抑制NF κB的激活 ,逆转耐药细胞对?Objective To study the sensitizing effects of a double stranded decoy oligodeoxynucleotides (ODNs) with a specific affinity for NF κB on hepatic cancer cells to chemotherapy. Methods After HepG 2 cells resisting to adriamycin (HepG 2/ADR) were induced stepwise, FITC labeled decoy ODNs against NF κB activity were transfected into HepG 2/ADR cells with LipofectAMINETM 2000. The localization of decoy DNA was showed by inverse fluorescent microscopand confocel and EMSA was performed to investigate the affinity of NF κB introduction of double stranded ODNs into the nuclei of HepG 2 cells. ADM (0 1 mg/L) was administered. The proliferation was observed by MTT assay and the apoptosis of cells was observed by flow cytometry and TUNEL. Results HepG 2/ADR was confirmed being resistant to ADM. One hour after the decoy ODNs was transfected, FITC labeled decoy ODNs against NF κB was detected in the nuclei of HepG 2/ADM cells. EMSA showed the increase in NF κB binding to the nucleus. After incubation with ADM (0 1 mg/L), it showed significant inhibitory effect on the growth of HepG 2/ADM. The percentage of apoptosis was increased as compared with the control at 24 h. Conclusions Combined treatment with decoy ODNs for NF κB can be a novel and attractive strategy to improve therapeutic outcome of human hepatocellular carcinoma.

关 键 词：NF-ΚB ADM HepG2 寡核苷酸 肝癌 耐药细胞株 转染 共聚焦显微镜 结合活性 荧光显微镜 

分 类 号：R735.7[医药卫生—肿瘤]