缺氧诱导因子-1αsiRNA增强耐药肝癌细胞的化疗敏感性  

Enhancing sensitivity of Hepatocarcinoma cells to chemotherapeutic drugs by small interference RNA of hypoxia inducible factor-1α

作　　者：朱虹[1] 罗顺峰[2] 张万广[2] 关剑[2] 张必翔[2] 陈孝平[2] 

机构地区：[1]苏州大学医学院基础医学系,215006 [2]华中科技大学同济医学院附属同济医院肝胆胰外科研究所

出　　处：《中华实验外科杂志》2007年第12期1514-1516,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(30371395)

摘　　要：目的观察缺氧诱导因子-1α(HIF-1α)特异性小分子干扰 RNA(siRNA)对肝癌化疗的增敏作用。方法构建 HIF-1αsiRNA 真核表达质粒,经脂质体稳定转染于耐药的 C28肝癌细胞。研究分为实验组、阴性对照组和空白对照组。荧光定量 PCR 和 Western blot 技术分别检测3组细胞中多药耐药相关基因 MDR1、LRP、MRP1在 mRNA 和蛋白水平的表达,PI 法流式细胞术分析3组细胞在受到5-Fu 作用后的凋亡指数。每组细胞分别随机注入10只裸鼠皮下,比较3组细胞成瘤后对 ADM 治疗的反应性。结果 HIF-1αsiRNA 能有效抑制 HIF-1α基因的表达,并能使 C28耐药肝癌细胞内 MDR1、MRP1、LRP 基因在 mRNA 和蛋白水平表达明显下降。在5-Fu 作用后第24、48、72小时,实验组细胞的凋亡指数分别是阴性对照组的2.88、3.56和3.87倍,实验组对化疗药物5-Fu 的敏感性显著增强(P<0.01)。注射阿霉素后,实验组裸鼠皮下的耐药肝癌瘤体明显缩小,肿瘤抑制率为41.35%,与阴性对照组比较差异有统计学意义(P<0.01)。结论成功构建了 HIF-1α-siRNA 的真核表达质粒。HIF-1α靶序列特异性的 siRNA 可增强肝癌细胞对化疗药物的敏感性,它与传统化疗药物的联合应用可望实现对肝癌的有效治疗。Objeαive To explore the effeα of specific small interference RNA of hypoxia inducible faαor-1α on enhancing sensitivity of hepatocellular carcinoma to chemotherapeutic drugs. Methods PSilencer2.1/HIF-1 α-siRNA eukaryotic expression plasmid was construαed. Liposome carrying the plasmid was stably transfeαed into C28 hepatocarcinoma cell line resistant to multiple chemotherapeutic drugs. The whole experiments were divided into three groups ： experiment group, negative control group and empty control group. Real-time fluorescent quantitative PCR and Western-blot technique were respeαively used to analyze the expression of MDR1, MRP1 and LRP at mRNA and protein level in the above three group cells. Flow cytometry was used to determine apoptosis index of those cells after being administrated by 5- Fu. Randomly,every group of cells was subcutaneously inoculated into 10 nude mice. After subcutaneous neoplasm having grown, their sensitivities to ADM were compared among these three groups. Results HIF-1α-siRNA could specifically inhibit HIF-1α expression. Moreover, HIF-1α-siRNA made the expression amounts of MDR1, MRP1, LRP at mRNA and protein level decrease in C28 hepatocarcinoma cell hne. At 24h ,48h and 72h after administration of 5-Fu, apoptosis index of the experiment group was respectively 2.88,3.56 and 3.87 folds of that of negative control group,which suggested the sensitivity of experiment group to 5-Fu was significantly increased. Average size of subcutaneous neoplasm of nude mice in experiment group was remarkably reduced after being treated by ADM and tumor inhibiting rate was 41.35% ,which was significantly different from negative control group （P 〈 0.01 ）. Conclusion HIF-1α- siRNA expressed plasmid is successfully construαed. SiRNA targeted HIF-1 α gene could enhance the sensitivity of hepatocareinoma cells to chemotherapeutic drugs. And an effeαive therapeutic strategy for hepa- tocellular carcinoma could possibly be realized by united administration of conventional chemotherapeutic a

关 键 词：小分子干扰RNA 缺氧诱导因子-1Α 抗药性 多药 癌 肝细胞 

分 类 号：R735.7[医药卫生—肿瘤]