无血清培养肾癌干细胞及其鉴定  

Isolation and identification of renal carcinoma stem cells in serum free medium

作　　者：潘鹏[1] 郭涛[1] 田福起[1] 孙浩[1] 马克钧[1] 徐强[1] 

机构地区：[1]江苏大学附属医院泌尿外科,江苏省镇江市212001

出　　处：《中国组织工程研究与临床康复》2009年第32期6284-6288,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：镇江市社会发展科技计划资助项目(SH2007024)~~

摘　　要：背景:肿瘤干细胞理论的产生,对肿瘤的发生、发展及其发病机制有了更新的认识,为肿瘤研究提供了新的方向,通过无血清培养技术已从许多肿瘤中分离并鉴定出特定的肿瘤干细胞。目前,无血清培养已成为培养肿瘤干细胞的经典培养方法。目的:运用无血清培养的方法从肾癌细胞中获取肾癌干细胞,并进行鉴定。设计、时间及地点:细胞学体外观察,于2008-02/2009-02在江苏大学完成。材料:肾癌细胞株OS-RC-2由上海中科院细胞库提供。方法:取处于对数生长期的肾癌细胞OS-RC-2,悬浮于预先配好的DMEM/F12无血清培养基中(含表皮生长因子20μg/L,碱性成纤维细胞生长因子20μg/L),观察生长情况,培养7d后运用流式细胞仪测定CD133及CD34的表达。以不含细胞因子的高糖DMEM/F12培养液培养细胞作为阴性对照。另外收集无血清培养7d的肾癌细胞球,重悬于含体积分数为10%胎牛血清的高糖DMEM/F12培养基中,进行干细胞分化实验观察。主要观察指标:①倒置显微镜下观察肾癌干细胞生长情况。②流式细胞仪检测肾癌干细胞及肾癌细胞中CD133及CD34的表达。③收集无血清培养7d后的肾癌干细胞重新常规培养并观察其分化情况。结果:①肾癌细胞接种于含细胞因子的无血清培养基2d后可见有细胞球生成,7d后细胞球明显增多,体积增大。对照组肾癌细胞则贴壁生长,未见细胞球生成。②流式细胞仪检测显示,肾癌细胞球中CD133+CD34-表达率(8.33±1.26)%,对照组肾癌细胞表达率(1.24±0.36)%(t=-19.71,P<0.05)。③肾癌细胞球重新悬浮于含体积分数为10%胎牛血清的培养液2d后可见细胞球分散为单个细胞,恢复原来贴壁生长特点,CD133及CD34表达与阴性对照相同。结论:利用含细胞因子表皮生长因子和碱性成纤维细胞生长因子无血清培养的方法可以从肾癌细胞中获取肾癌干细胞。BACKGROUND： The proposition of renal cell carcinoma （RCC） stem cell theory put forward the study underlying genesis,development and pathogenesis of tumor, which provide a newly approach to tumor research. Specific tumor stem cell has been separated and identified with serum-free culture. Presently, serum-free has been the classical method for tumor stem cell culture. OBJECTIVE： To obtain renal cell carcinoma （RCC） cells line with serum free medium （SFM） culture, in addition, to identify the cells. DESIGN, TIME AND SETTING： The in vitro cytology experiment was conducted at the Jiangsu University between February 2008 and February 2009. MATERIALS： RCC cells line OS-RC-2 was provided by Cell Bank of Chinese Academy of Sciences. METHODS： RCC cells was collected in logarithmic growth phase and dissociated into single cell, then they was seeded in DMEM/F12 serum free medium containing 20μg/L epidermal growth factor （EGF）, and 20μg/L basic fibroblast growth factor （bFGF）. After generation, the growing process of cells was observed, and the expression of CD133 and CD34 in cells from SFM was detected by flow cytometry at days 7 after culture. RCC cells seeded with serum free DMEM/F12 medium were served as control group. At last the cells from SFM were recultivated in 10% fetal bovine serum （FBS） medium, and the growth state was observed. MAIN OUTCOME MEASURES： The growth of RCC stem cells was observed by an inverted microscopy. The expression of CD133 and CD34 was detected by flow cytometry. Meantime, differentiation of conventional cultured RCC cells after 7 days of SFM culture was observed. RESULTS： ①The RCC cell spheres formed at 2 days after culture in SFM with cell factors, and the spheres volume became larger and their numbers became increased after 7 days. In the control group, RCC cells grew adhesion to walls without cell spheres formed.②The flow cytometry revealed that expressions of CD133^＋CD34^- were （1.24±0.36）% in RCC cells, which reached to （8.33±1.26）%

关 键 词：肿瘤干细胞 肾癌 细胞培养 CD133 CD34 

分 类 号：R394.2[医药卫生—医学遗传学]