无血清悬浮培养肾癌干细胞表面标志CD133及端粒酶活性的表达

CD133 expression and telomerase activity in serum-free cultured renal carcinoma stem cells

背景：有研究表明端粒酶活性抑制剂不仅能抑制或杀死肾癌细胞,而且对决定肾癌发生发展的干细胞也有作用。目的：观察无血清悬浮培养的肾癌干细胞表面标志CD133及端粒酶活性的表达情况,并与含血清培养的肾癌细胞作比较。设计、时间及地点：细胞学体外观察,于2008-06/2009-02在江苏大学完成。材料：手术切除肾癌周围新鲜的正常肾组织标本由江苏大学附属医院提供,肾癌干细胞株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干细胞OS-RC-2,胰酶消化后离心弃上清,悬浮于含EGF、bFGF的DMEM/F12无血清培养基中,调整细胞浓度为2×10^8L-1进行接种,在37℃、体积分数为5%的CO2培养箱中悬浮培养。以含血清培养的肾癌细胞及正常肾组织细胞作为对照。主要观察指标：倒置显微镜下观察细胞生长情况,流式细胞仪检测肾癌干细胞CD133及CD34的表达,采用TRAP实时定量检测肾癌干细胞端粒酶活性。结果：肾癌干细胞接种于无血清培养基后,细胞呈圆形悬浮于培养液中;2d后有细胞球生成,每个细胞球含3~8个细胞,折光性较强;7d后细胞球明显增多,体积增大,为规则的圆形或卵圆形。无血清悬浮培养的肾癌干细胞球CD133＋CD34-率明显高于含血清培养的肾癌细胞CD133＋CD34-率,正常肾组织细胞未测出有CD133及CD34的表达,3者间比较差异有显著性意义（F=328.25,P〈0.05）。肾癌干细胞球和肾癌细胞端粒酶活性均高于正常肾组织细胞（F=278.74,P〈0.05）,而前2者间端粒酶活性无明显差异。结论：与含血清培养的肾癌细胞相比,无血清悬浮培养的肾癌干细胞表面标志CD133呈明显高表达,二者端粒酶活性均高于正常肾组织。

BACKGROUND： Telomerase activity inhibitor inhibits or kills renal carcinoma cells, and also affects stem cells that play importan roles in occurrence and development of renal carcinoma. OBJECTIVE： To observe renal carcinoma stem cell surface marker CD133 and telomerase activity expression in serum-free suspension culture, and to compare with renal carcinoma cells in serum suspension culture. DESIGN, TIME AND SETTING： The in vitro cytological study was performed at the Jiangsu University from June 2008 to February 2009. MATERIALS： Fresh normal renal tissue surrounding renal carcinoma was obtained from Affiliated Hospital, Jiangsu University. Renal carcinoma stem cell line OS-RC-2 was supplied by Cell Bank, Chinese Academy of Sciences Shanghai Branch. METHODS： OS-RC-2 in Iogadthmic phase, digested by trypsin, and centdfuged. Supernatant was removed. OS-RC-2 cell line in serum-free DMEM/F12 supplemented with epidermal growth factor and basic fibroblast growth factor was incubated at 2×10^8/L in 5% CO2 incubator at 37℃. Renal carcinoma cultured in serum and normal renal tissue served as controls. MAIN OUTCOME MEASURES： Cell growth was observed under an inverted microscope. Expression of CD133 and CD34 was detected using flow cytometry. Real-time quantitative TRAP assay was applied to evaluate telomerase activity in renal carcinoma stem cells. RESULTS： After incubated in serum-free medium, renal carcinoma stem cells were round and suspended. Two days later, cell mass generated. Each cell mass contained 3-8 cells, with strong refraction. Seven days later, cell mass became more, presented big body that was regular, round or elliptical. CD133＋CD34 rate in renal carcinoma stem cell mass was significantly greater in serum-free suspension culture compared with in serum suspension culture. CD133 and CD34 expression was not determined in normal renal tissue. There were significant differences among groups （F=328.25, P 〈 0.05）. Telomerase activity was greater in renal carcinoma stem cells and renal carcinoma cells compared with normal renal cells （F=278.74, P 〈 0.05）. No significant difference was detected between renal carcinoma stem cells and renal carcinoma cells. CONCLUSION： Compared with serum cultured renal carcinoma cells, serum-free cultured renal carcinoma cell surface marker CD133 presents high expression. Moreover, telomerase activity is high in renal carcinoma stern cells and renal carcinoma cells compared with normal renal tissue.