肾癌中CD133~+细胞和CD133~-细胞生物学特性差异的研究
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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G_0/G_1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)%,G_2/M分别为(10.83±0.35)%、(12.24±0.31)%,OS-RC-2中CD133~+细胞、CD133~-细胞G_0/G_1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)%,G_2/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。
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 G_0/G_1 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 G_2/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.
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G_0/G_1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)%,G_2/M分别为(10.83±0.35)%、(12.24±0.31)%,OS-RC-2中CD133~+细胞、CD133~-细胞G_0/G_1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)%,G_2/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。
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 G_0/G_1 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 G_2/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.
引文
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