缺氧对人乳腺癌细胞微球体生长及化疗耐药性的影响.doc

缺氧对人乳腺癌细胞微球体生长及化疗耐药性的影响 范原铭，屈洪波，吴诚义，侯 婧，韩明利 （400016 重庆，重庆医科大学附属第一医院内分泌乳腺外科） [摘要目的研究缺氧对人乳腺癌细胞微球体（MSs）培养效率其化疗耐药性的影响。方法 分别在常氧及缺氧条件下对人乳腺癌MDA-MB-231细胞进行无血清悬浮培养，观察记录MSs的生长速率及体积变化。MTT比色法检测各组人乳腺癌微球体细胞（mammospheres-derived cells，MSDCs）及MDA-MB-231细胞在不同浓度、不同药物作用下的存活率（urvive Rate，SR）。免疫组织荧光化学法检测HIF-2α、ABCG2在各组MSDCs中的表达。Western blot检测乳腺癌干细胞标志物CD44及ALDH1在各组MSDCs中的表达。Western blot及Real-time RT-PCR检测HIF-2α、ABCG2、P-g及MDR1在各组MSDCs中的表达。结果 缺氧组MSs生长速率及球体体积优于常氧组。缺氧组MSDCs在不同化疗药物作用下的SR高于常氧组MSDCs及MDA-MB-231细胞＜0.05。免疫组织荧光化学检测发现HIF-2α及ABCG2在缺氧组MSDCs中的表达强于常氧组。Western blot检测发现CD44与ALDH1在缺氧组MSDCs中表达量高于常氧组。缺氧组MSDCs中HIF-2α、ABCG2及P-gP蛋白表达量均高于常氧组MSDCs及MDA-MB-231细胞中的表达＜0.05。Real-time RT-PCR检测发现缺氧组MSDCs中HIF-2α、ABCG2及MDR1 mRNA的表达量均高于常氧组MSDCs及MDA-MB-231细胞＜0.05。结论 以HIF-2α为启动因子，ABCG2、MDR1及P-g等为效应因子的生物化学反应可能是缺氧状态加快MSs的成球速度提高其球体体积增强其化疗耐药能力的可能机制。缺氧诱导因子2α重庆市医学科研计划项目06-2-028） ［通信作者吴诚义E-mail：wuchengyicq@163.comStudy of the influence of hypoxia on human breast cancer mammospheres culture and resistance to chemotherapy Fan Yuanming1,Qu Hongbo1,Wu Chengyi1,Dong Ning2,Hou Jing1,Han Mingli1 （1Department of Endocrine Breast Surgery, First Affiliated Hospital, Chongqing Medical University, Chongqing ,400016;2Department of General Surgery，The people’s Hospital of Changshou District, 401220,China） [Abstract Objective] To study the effect of hypoxia on human breast cancer cell microspheres’ culture and resistance to chemotherapy, and discuss the related mechanisms. Methods: MDA-MB-231 cells were cultured in the normal oxygen and hypoxia serum-free media supplemented with growth factors. The culture times and sizes of the MSs were recorded. The survival rates of each group MSs and MDA-MB-231 cells under various chemotherapy drugs with various concentrations were detected by MTT colorimetric method .The expressions of HIF-2α and ABCG2 in each group MSs were observed by Immunofluorescence technique. The expressions of CD44 and ABCG2 of each group were detected by Western blotting. The expressions of HIF-2α, ABCG2,