下调XIAP表达增强化疗药物诱导胃癌细胞凋亡的作用  

Effects of down-regulating expression of X-linked inhibitor of apoptosis on chemotherapyinduced apoptosis of gastric cancer cells

作　　者：郑丽端[1] 童强松[2] 汪良[2] 董继华[3] 侯晓华[4] 

机构地区：[1]华中科技大学同济医学院附属协和医院病理科,湖北省武汉市430022 [2]华中科技大学同济医学院附属协和医院外科,湖北省武汉市430022 [3]华中科技大学同济医学院附属协和医院中心实验室,湖北省武汉市430022 [4]华中科技大学同济医学院附属协和医院消化内科,湖北省武汉市430022

出　　处：《世界华人消化杂志》2007年第29期3067-3073,共7页World Chinese Journal of Digestology

基　　金：国家自然科学基金;No.30600278;教育部新世纪优秀人才支持计划资助项目;No.NCET-06-0641

摘　　要：目的:观察下调X连锁凋亡抑制蛋白(XIAP)基因表达对胃癌细胞化疗敏感性的影响.方法:构建XIAP基因反义真核表达载体,稳定转染胃癌细胞株MKN-45,RT-PCR和Western blot法检测癌细胞XIAP基因表达.选用顺铂、丝裂霉素分别处理转染前后的胃癌细胞,采用MTT比色法、克隆形成抑制实验检测癌细胞体外生长活性:透射电镜、流式细胞术、TUNEL检测癌细胞凋亡及比率;Western blot和比色法检测细胞内caspase-3蛋白表达和活性水平.结果:RT-PCR和Western blot证实,稳定转染反义XIAP基因的胃癌细胞MKN-45的XIAP mRNA和蛋白表达水平分别降低84.75%(P<0.01)和89.75%(P<0.01),各浓度顺铂、丝裂霉素处理24 h后,转染反义XIAP基因的MKN-45细胞生长抑制率分别增加7.3%-25.3%(P<0.01),12.3%-16.3%(P<0.01).透射电镜下可见部分细胞发生典型的凋亡形态学改变,凋亡率分别为34.12%和32.5%,显著高于未转染对照组MKN-45细胞的凋亡率(14.2%,P<0.05).与MKN-45细胞比较,稳定转染反义XIAP基因的MKN-45细胞内caspase-3表达水平增高2.45倍(P<0.01),活性水平提高3.68倍(P<0.0 1).结论:通过反义RNA技术下调XIAP基因表达,能提高癌细胞中caspase-3的表达和活性,增强化疗药物对癌细胞的诱导凋亡作用.AIM： To observe the effects of down-regulating expression of X-linked inhibitor of apoptosis （XIAP） on the chemotherapeutic sensitivity of gastric cancer cells METHODS： The antisense eukaryotic vector for XIAP was constructed and stably transfected into gastric cancer cell line MKN-45. Reverse transcription polymerase chain reaction （RTPCR） and Western blotting were applied to detect XIAP gene expression. Cisplatin and mitomycin C were administrated to untransfected and transfected gastric cancer cells. MTT colorimetry and clone formation were performed to measure in vitro cell viability. Apoptosis and its rates were detected by electron microscopy, acridine orange-ethidium bromide fluorescent staining and in situ terminally labeled transferase technique （TUNEL）. Cellular caspase 3 protein expression and its activity were assayed by Western blotting and colorimetry. RESULTS： RT-PCR and Western blotting indicated that the mRNA and protein levels within gastric cancer MKN-45 cells stably transfected with antisense XIAP vector were significantly decreased by 84.75% （P 〈 0.01） and 89.75% （P 〈 0.01）, respectively. After treatment with various concentrations of cisplatin and mitomycin C for 24 h, growth inhibition of MKN-45 cells stably transfected with antisense XIAP was enhanced by 7.3%-25.3% （P 〈 0.01） and 12.3%-16.3% （P 〈 0.01）, respectively. Partial cancer cells presented characteristic changes of apoptosis under electron microscopey, with apoptosis rates of 34.12% and 32.5%, respectively, which were significantly higher than those in untransfected control MKN-45 cells （14.2%, P 〈 0.05）. Compared with MKN-45 cells, caspase 3 expression in cells stably transfected with antisense XIAP was significantly increased by 2.45 times （P 〈 0.01）, while the activity of caspase 3 was enhanced by 3.68 times （P 〈 0.01）. CONCLUSION： Down-regulation of XIAP expression via antisense RNA may increase the expression and activity of caspase 3 and enhance chemotherapy-induce

关 键 词：胃癌 XIAP基因 反义RNA 基因表达 细胞凋亡 

分 类 号：R735.2[医药卫生—肿瘤]