肾癌OSRC2细胞CD133表达及意义.docVIP

肾癌OSRC2细胞CD133表达及意义 　 作者：田福起 孙浩, 潘鹏, 郭涛 【摘要】 目的： 培养肾癌干细胞球，研究其表面分子特性。方法： 肾癌细胞悬浮于含有表皮生长因子(epidermal growth factor, EGF)和成纤维生长因子(basic fibroblast growth factor, bFGF)的无血清培养基中培养，培养7天后收集细胞球，流式细胞仪检测CD133及CD34的表达。结果： 悬浮培养2天后出现肾癌干细胞球，培养7天后干细胞球明显增多，流式细胞仪检测发现表达CD133+CD34-细胞约占(8.33±1.26)%，对照肾癌细胞组表达CD133+CD34-细胞只占(1.24±0.36)%;干细胞球通过含10%胎牛血清培养基继续培养，又恢复原来的生长特点。结论： 利用无血清悬浮培养方法得到的肾癌干细胞球高表达干细胞表面分子标记CD133。 【关键词】 肿瘤干细胞; 肾癌; 细胞培养; CD133; CD34 　　[Abstract] Objective: To cultivate renal cell carcinoma(RCC) stem cells in serum free medium(SFM) with the method of suspension culture and to research their property of surface molecules on the cells.Methods： RCC cells were collected and dissociated in to single ones, then they were seeded in SFM containing EGF and bFGF. Seven days later the expression of CD133 and CD34 in cells from SFM was detected by flow cytometry. Results： RCC stem cells spheres were appeared after RCC cells had been cultured two days in SFM ; Seven days later, more spheres came out . Then collected and detected them by flow cytometry. We found that about(1.24±0.36)%CD133+CD34-cells were in normal OSRC2 cell line, while it reached to (8.33±1.26)% after cultivating in SFM for 7 days. After the cell spheres were recultivated in SSM, CD133+CD34-cells became less and close to before. Conclusion： RCC stem cells spheres enriched from RCC cells by SFM had higher expression of CD133. 　　[Key words] tumor stem cell; renal cell carcinoma(RCC); cell culture; CD133; CD34 　　肿瘤干细胞的鉴定主要利用细胞特异表面标志及侧群(side population, SP)检测。目前已相继从白血病、乳腺癌、脑胶质瘤等多种肿瘤组织中得到了相应的肿瘤干细胞。本文旨在运用无血清培养基培养肾癌细胞，流式细胞仪鉴定其CD133，CD34的表达，明确肾癌干细胞的表面标志。 　　1 材料与方法 　　1.1 仪器与试剂 　　CO2培养箱(上海力康)、流式细胞分析仪(BD公司)、超净工作台(苏州安泰)、Anke TDL5A离心机(上海安亭);肾癌细胞株OSRC2(上海中科院细胞库);DMEM高糖培养液(GBICO)、F12培养液(GBICO)、胎牛血清(杭州四季青)、bFGF(Prospec)、EGF(Prospec)、antiCD133PE(eBioscience)、antiCD34-FITC (Sigma)。 　　1.2 方 法 　　1.2.1 肾癌干细胞球的培养 取对数生长期的肾癌细胞OSRC2，用0.25%的胰酶消化，倒置显微镜下观察，待细胞由梭形变为圆形时加入含10%胎牛血清的高糖DMEM培养液终止消化;收集细胞，1 5