【作者】 宋峻岩；

【导师】 岳中瑾；

【作者基本信息】 兰州大学 ， 外科学， 2008， 硕士

【摘要】 目的:人CD133（Human prominin-1 PROM-1）是在不同组织中鉴定干/祖细胞或肿瘤干细胞（肿瘤启动细胞）（cancer stem cells,tumor-initiating cells）的标志物。本文目的在于研究对比人肾癌细胞系786-0和OS-RC-2中CD133⁺细胞和CD133^-细胞的生物学特性差异。方法:应用流式细胞仪检测肾癌细胞系786-0和OS-RC-2中CD133的膜表达状况;以免疫磁珠法将CD133⁺细胞和CD133^-细胞分离纯化,比较两者在光镜下的形态、生长特点、体外增殖能力、长期分化能力及体外克隆形成率的差异;流式细胞术分析两者细胞周期分布的变化,采用MTT法比较两者对化疗药物顺铂（DDP）敏感性的差别。结果:786-0和OS-RC-2中均有CD133的少量表达（8.12±0.86）%、（6.83±0.20）%,CD133⁺细胞相比CD133^-细胞具有较强的体外增殖、克隆形成及长期分化能力;786-0中CD133⁺细胞、CD133^-细胞以及未分选细胞群体倍增时间分别为19.46h、22.03h、20.03h:OS-RC-2中CD133⁺细胞、CD133^-细胞以及未分选细胞群体倍增时间分别为18.84h、22.26h、21.18h;786-0和OS-RC-2中CD133⁺细胞克隆形成率分别为（38.47±4.27）%和（32.47±5.21）%,CD133^-细胞克隆形成率分别为（21.38±3.58）%和（26.04±7.12）%;786-0中CD133⁺细胞、CD133^-细胞G₀/G₁期分别为（65.77±0.81）%、（44.9±2.21）%,S期分别为（13.65±0.42）%、（39.66±1.25）%,G₂/M分别为（10.83±0.35）%、（12.24±0.31）%,OS-RC-2中CD133⁺细胞、CD133^-细胞G₀/G₁期分别为（61.58±1.32）%、（42.3±3.11）%,S期分别为（12.17±0.22）%、（37.80±0.32）%,G₂/M期分别为（11.36±0.22）%、（16.23±0.36）%,CD133⁺细胞、CD133^-细胞两者相比差异均有显著性统计学意义（P＜0.01）。MTT法分析表明CD133⁺细胞、CD133^-细胞对DDP敏感性差异无统计学意义。结论:证实肾癌细胞系中CD133⁺细胞具有肾癌干细胞的部分特征,能否作为肾癌干细胞的表面标志还需要进一步的实验加以明确。更多还原

【Abstract】 Background CD133 was used as a marker to detect stem cells （progenitor cells）and cancer stem cells（tumor-initiating cells）in various tissues.The purpose of this study was to investigate the different biological characteristics of CD133⁺ cells and CD133^-cells in renal cell carcinoma with in vitro analyses.methods The CD133 surface expression of both 786-0 and OS-RC-2 cell lines was evaluated by flow cytometry.CD133⁺ cells were isolated by MACS（magnetic activated cell sorting）and examimed in vitro proliferation,growth characteristics and colony formation efficiency（CFE）. Flow cytometry was used to evaluate the cell cycle distribution and long-term differentiation ability.The sensitivity to diamminedichloroplatinum（DDP）between CD133⁺ cells and CD133^-cells were contrasted by drug sensitivity testing in vitro（MTT assay）.Result CD133 surface expression were found in 2 /2 RCC cell lines.It was shown that isolated CD133⁺ cells from 786-0 and OS-RC-2 cell lines are more proliferetive and have higher colony-forming and more long-term differentiation abilities than CD133^-cells in vitro.CD133⁺ cells from 786-0 and OS-RC-2 cell lines had relatively shorter colony doubling time than CD133^-cells[（19.46 h vs 22.03 h）and（18.80 h vs 22.26 h）] indicating that the growth of CD133⁺ cells was faster.There was significant difference in the mean CFE between CD133⁺ cells and CD133^-cells of 786-0 and OS-RC-2 cell lines[（38.47±4.27）%vs（21.38±3.58）%and （32.47±5.21）%vs（26.04±7.12）%,P＜0.01].There also was significant difference in the cell cycle distribution between CD133⁺ cells and CD133^-cells[（65.77±0.81）%vs（44.9±2.21）%and（61.58±1.32）%vs （42.93±3.11）%]in G₀/G₁ phase,（13.65±0.42）%vs（39.66±1.25） %and（12.17±0.22）%vs（37.80±0.32）in S phase,and（10.83±0. 35）%vs（12.24±0.31）%and（11.36±0.22）%vs（16.23±0.36）% in G₂/M phase,P＜0.01].Additionally,the difference of sensitivity to diamminedichloroplatinum（DDP）between CD133⁺ cells and CD133^-cells was not significant.Conclusion It was confirmed that CD133⁺ cells in RCC cell lines are useful markers for the detection of cancer stem cells（tumor-initiating cells）.The subsequent further biological characterization of CD133⁺ cells in RCC cell lines shoul reveal whether they possess features of stem cells such as the capacity for increased tumorgenity.The biological characteristics of CD133⁺ cells in RCC cell lines will help elucidate more details of cancer stem cells in RCC.更多还原