论文部分内容阅读
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: The cancer stem cell hypothesis suggests that tumor clonal growth is maintained by a small proportion of stem cell-specific cells in tumor tissue. In recent years, several solid tumor tumor stem cells have been isolated one after another, but so far no confirmed objective confirmation of bladder cancer stem cells and its separation and identification of effective methods reported. Objective: To verify the possibility of bladder cancer stem cells and the possibility of EMA as a surface marker of bladder cancer stem cells. Design: observe the contrast experiment. Unit: Tianjin Institute of Urology, Tianjin Medical University Second Hospital. MATERIALS: Experiments were performed at the Tumor Immunology Laboratory of Tianjin Institute of Urology from March 2006 to July 2007 at Key Laboratory of Biomedical Engineering. Nine cases of bladder tissue samples from the Second Hospital of Tianjin Medical University, in line with low-potential malignant bladder papillary urothelial tumors and low-grade papillary urothelial carcinoma diagnostic criteria. Another 40 cases of low-grade malignant transitional cell carcinoma of the bladder and 10 cases of normal bladder epithelium for immunohistochemistry were from the Department of Urology, the Second Hospital of Tianjin Medical University. Informed consent was obtained before the specimens were taken. Methods: ①Through DNA chip technique to compare the difference of normal urothelial and bladder cancer gene expression to determine the possibility of bladder cancer stem cells. ② Immunohistochemistry was used to detect the expression of Bmi-1 and EZH2 in low grade bladder transitional cell carcinoma. ③ Observe the positive localization of 27 potential markers in normal and low-grade bladder transitional cell carcinoma tissues. (4) The EMA-subpopulations were obtained by immunomagnetic bead cell sorting system and their clonogenicity, self-renewal and proliferation ability were analyzed. MAIN OUTCOME MEASURES: ① The difference of gene expression between normal urothelial and bladder cancer. ② The expression of Bmi-1 and EZH2 in low grade bladder transitional cell carcinoma and normal bladder epithelium. ③ cloning formation of experimental results. Results: ①A total of 268 differentially expressed genes (including Bmi-1 and EZH2) were found, and Bmi-1 and EZH2 overexpressed in low-grade bladder transitional cell carcinoma. ② In addition to EMA, 26 other possible bladder cancer stem cell surface markers have no practical value in the isolation of bladder cancer stem cells. ③EMA - is located in the normal urothelial and bladder cancer tissues basal cell layer (possible stem cell location), EMA - subpopulation with clonogenic, self-renewal and proliferation ability. Conclusion: The experiment confirmed the existence of bladder cancer stem cells, EMA- may be its surface marker.