目的脑肿瘤耐药是否与其干细胞相关和耐药机制何在还不清楚,本实验旨在探讨脑肿瘤干细胞耐药的分子机制。方法从人脑胶质瘤组织标本中分离CD133+细胞,在无血清条件下体外长期传代培养,取其中呈悬浮生长的细胞球,再用CD133免疫磁珠分离其中的阳性细胞在无血清条件下培养。将尼卡地平、米托蒽醌分别或联合作用于上述的培养细胞,观察细胞形态、增殖抑制和细胞凋亡率等变化。结果经CD133免疫磁珠筛选过的细胞球在相差显微镜下观察到尼卡地平与米托蒽醌联合作用组细胞的细胞毒性非常明显,有的球体已崩解,增殖抑制很明显,并且对米托蒽醌呈浓度依赖性,在2.5或5.0μmol·L-1的尼卡地平协同下的米托蒽醌,于10-6～10μmol·L-1时对肿瘤细胞的增殖抑制呈浓度依赖性与空白对照和单纯尼卡地平组相比均有统计学差异(P<0.01);而对未经CD133免疫磁珠筛选球体的抑制作用不显著。流式细胞仪检测表明,尼卡地平能协同米托蒽醌促进受试细胞的凋亡。结论脑肿瘤干细胞高表达ABCG2是其耐化疗药物的原因之一,尼卡地平通过竞争性地抑制ABCG2作用而提高化疗药物的敏感性。
Abstract
OBJECTIVE To invesgigate the molecular mechanisms of drug resistance of brain tumor stem cells.METHODS CD133+ brain tumor stem cells were isolated and identified from human glioma tissue.Cells were cultured in DMEM/F12 medium containing growth factors.Cell spheres were sorted by CD133 cell isolation kit.CD133+ brain tumor stem cells were treated with nicardipine and/or mitoxantone.Cell morphology,anti-proliferative effect and apoptosis on human glioma cell lines were analyzed. RESULTS Compared with control treatment with blank solution or mitoxantone alone,the proliferation of CD133+ brain tumor stem cells were remarkably inhibited by nicardipine(2.5 or 5.0μmol·L-1) and mitoxantone(10-6-10μmol·L-1).However,treatment with nicardipine and mitoxantone had no effect on inhibition of proliferation in brain tumor stem cell spheres which did not sort by CD133 isolation kit.Results of flow cytometry indicated the improving effect of coordination of nicardipine and mitoxantone on apoptosis of brain tumor stem cells,CONCLUSION Over-expression of ABCG2 is one of the molecular mechanisms of drug resistance of brain tumor stem cells.Nicardipine improved durg sensitivity of brain tumor stem cells by competitive inhibition on ABCG2.
关键词
脑肿瘤干细胞 /
ABCG2基因 /
肿瘤耐药 /
尼卡地平 /
米托蒽醌
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Key words
brain tumor stem cells /
ABCG2 /
drug resistance /
nicardipine /
mitoxantone
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脚注
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基金
国家自然科学基金资助项目(30400457,30672164)
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