摘要
背景与目的:细胞凋亡异常是肿瘤细胞产生耐药性的关键因素之一。Smac(secondmitochondria-derivedactivatorofcaspases,Smac或称DIABLO)是新近发现的一种凋亡调节基因,在介导化疗药物诱导肿瘤细胞凋亡中起重要作用。本研究旨在观察Smac基因过表达对胃癌细胞株化疗敏感性的影响。方法:采用脂质体GeneSHUTTLE-40介导的方法,将Smac基因转入胃癌细胞MKN-45,RT-PCR和Westernblot法检测癌细胞中Smac的表达;选用顺铂(1、5、10μg/ml)、丝裂霉素(0.1、1、10μg/ml)和姜黄素(10、20、40μmol/L)分别处理转染前后的胃癌细胞,四甲基偶氮唑蓝(MTT)比色法检测细胞增殖活性,倒置显微镜下观察细胞形态变化并摄影,AnnexinV-FITC和碘化丙啶双染色,流式细胞仪检测细胞凋亡。结果:同未转染对照组比较,转染外源性Smac基因后MKN-45细胞SmacmRNA和蛋白表达水平显著增高(P<0.01);各浓度顺铂、丝裂霉素和姜黄素处理24h后,细胞生长抑制率分别增加10.10%～23.80%(P<0.01)、10.01%～15.86%(P<0.01)、11.28%～22.12%(P<0.01),细胞明显变圆、折光增强、漂浮细胞增多,细胞凋亡率分别增加6.7%～20.2%(P<0.01)、5.4%～13.2%(P<0.01)、10.6%～20.1%(P<0.01)。结论:转染外源性Smac基因并使其在胃癌细胞中过表达。
BACKGROUND & OBJECTIVE: Abnormal apoptosis is one of the key fa ctors for drug resistance of neoplasms. Smac, a novel gene involved in regulatio n of apoptosis,plays an important role in tumor cell apoptosis induced by chemot herapeutic drugs. This study was designed to explore the effects of overexpresse d Smac gene on chemotherapeutic sensitivity of gastric cancer cell line. METHODS : By liposome GeneSHUTTLE- 40, Smac gene was transfected into gastric cancer ce ll line MKN- 45.Cellular Smac gene expression were determined by reverse transc ription- polymerase chain reaction (RT- PCR)and Western blot analysis. Cisplat in (1,5,10 μ g/ml), mitomycin C (0.1,1,10 μ g/ml), and curcumin (10,20,40 μ m ol/L) were selected to treat untransfected and transfected MKN- 45 cells. Cellu lar proliferation activities were assayed by tetrazolium bromide (MTT) colorimet ry. The morphological changes of cancer cells were observed under inverted micro scope. Cellular apoptosis was determined by Annexin V- FITC and propidium iodid e staining flow cytometry. RESULTS: Compared with untransfected control group,Sm ac mRNA and protein levels in transfected MKN- 45 cells were significantly incr eased (P< 0.01). The growth inhibition rates of MKN- 45 cells treated with vari ous concentration of cisplatin,mitomycin C,and curcumin on MKN- 45 cells were i ncreased by 10.10% - 23.80% (P< 0.01), 10.01% - 15.86% (P< 0.01),11.28% - 22.12% (P< 0.01),respectively,with the cells becoming round,obviously refr acted,and fragmented. The cellular apoptosis rates were increased by 6.7 % - 20.2% (P< 0.01),5.4% - 13.2% (P< 0.01),and 10.6% - 20.1% (P< 0.01) ,respectively. CONCLUSION:Transfection of extrinsic Smac gene results in its ove r- expression in MKN- 45 cells,which could increase the chemotherapeutic sensi tivity of MKN- 45 cells.
出处
《癌症》
SCIE
CAS
CSCD
北大核心
2004年第4期361-366,共6页
Chinese Journal of Cancer
