人膀胱癌干细胞存在于EMA-细胞亚群(英文)

Human bladder cancer stem cells exist in epithelial membrane antigen-subset

背景:肿瘤干细胞假说认为肿瘤克隆性生长由肿瘤组织中一小部分具有干细胞特性的细胞维持。近年来几种实体瘤肿瘤干细胞陆续被分离出来,但至今未发现证实膀胱癌干细胞客观存在及其分离和鉴定有效方法的报道。目的:验证膀胱癌干细胞存在的可能性及EMA作为膀胱癌干细胞表面标志的可能性。设计:观察对比实验。单位:天津泌尿外科研究所,天津医科大学第二医院。材料:实验于2006-03/2007-07在国家"二一一"工程重点实验室天津市泌尿外科研究所肿瘤免疫室完成。9例膀胱组织标本来源于天津医科大学第二医院,符合低恶潜能膀胱乳头状泌尿上皮肿瘤及低分级乳头状泌尿上皮癌的诊断标准。另外40例用于免疫组织化学实验的低恶性膀胱移行上皮细胞癌及10例正常膀胱上皮均来自天津医科大学第二医院泌尿外科。留取标本前均签署知情同意书。方法:①通过DNA芯片技术比较正常泌尿上皮和膀胱癌基因表达的差异,以初步判断膀胱癌干细胞存在的可能性。②应用免疫组织化学方法检测干细胞相关基因Bmi-1和EZH2在低恶性膀胱移行上皮细胞癌组织中的表达情况。③观察27个潜在标志在正常和低恶性膀胱移行上皮细胞癌组织中的阳性定位情况。④通过免疫磁珠细胞分选系统获得EMA-亚群,分析其克隆形成、自我更新和增殖能力。主要观察指标:①正常泌尿上皮和膀胱癌基因表达的差异。②Bmi-1及EZH2蛋白在低度恶性膀胱移行上皮细胞癌与正常膀胱上皮中的表达差异。③克隆形成实验结果。结果:①发现了268个差异表达基因(包括Bmi-1和EZH2),Bmi-1和EZH2过表达于低度恶性膀胱移行上皮细胞癌。②除了EMA,其他26种可能的膀胱癌干细胞表面标志在分离膀胱癌干细胞方面没有应用价值。③EMA-位于正常泌尿上皮和膀胱癌癌组织的基底细胞层(可能的干细胞位置),EMA-亚群具有克隆形成、自我更新和增殖能力。结论:实验肯定了膀胱癌干细胞的存在,EMA-可能是它的表面标志物。

BACKGROUND： Cancer stem cell （CSC） hypothesis suggests that tumorous clones are maintained by a rare fraction of cells with stem cell proprieties. Several kinds of CSCs of solid tumor have been isolated in recent years. However, there have been fewer studies on the objective existence of bladder cancer stem cells （BCSCs） and on the methods to effectively isolate and identify BCSCs. OBJECTIVE： To investigate possibilities of BCSC existence and of epithelial membrane antigen （EMA） used as a surface marker of BCSC. DESIGN： A control observation experiment. SETTING： Tianjin Institute of Urinary Surgery ＆ Second Hospital of Tianjin Medical University. MATERIALS： This study was performed at the Room for Tumor Immunity of Tianjin Institute of Urinary Surgery （key laboratory for State ＂211 Project＂） from March 2006 to July 2007. Nine specimens of human bladder were obtained from patients who received treatment in the Second Hospital of Tianjin Medical University. These specimens corresponded to the diagnostic criteria of low malignant potential papillary urothelial neoplasm and low-grade papillary urothelial carcinoma. Additionally, 40 samples of human low malignant bladder transitional cell carcinomas （BTCC） and 10 samples of normal urothelium that were used for immunohistochemistry were obtained from the patients who received treatment in the Department of Urinary Surgery, Second Hospital of Tianjin Medical University. Written informed consent for the specimen providing was obtained from the patients, and the protocol was approved by the hospital＇s Ethics Committee. METHODS： The genes that were differentially expressed between normal urothelium and BTCC were identified through a DNA array assay to preliminarily determine the existence of BTCC. Overpressed stem cell related genes, Bmi- 1 and EZH2, were verified by immunohistochemistry. A total of 27 potential surface markers of BCSCs were assayed to determine the location of positive cells. EMA subsets were obtained through an immunomagnetic bead cell sorting system to analyze their abilities for colony forming, self-renewal and extensive proliferation. MAIN OUTCOME MEASURES： Normal urothelium and BTCC gene expression difference, Bmi-1 and EZH2 protein expression difference between low malignant BTCC and normal urothelium; and experimental results of colony forming. RESULTS： A total of 268 genes （including Bmi-1 and EZH2） that were differentially expressed between normal urothelium and low malignant BTCC were identified through a DNA array assay. The Bmi-1 and EZH2 had been found overexpressed in the low malignant BTCC. Except for EMA, above-mentioned 26 out of 27 potential surface markers were null for isolating BCSCs. EMA subsets were located in the basal layer and suprabasal epithelial layers of both normal and tumorous urothelium （potential location of BCSCs）. EMA subsets possesed the abilities for colony forming, self-renewal and extensive proliferation. CONCLUSION： The experiment confirms the existence of BCSCs. EMA might be a surface marker of BCSCs.