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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。
Whether the purpose of brain tumor resistance is related to its stem cells and the mechanism of drug resistance is unclear. This experiment aims to explore the molecular mechanisms of brain cancer stem cell resistance. Methods CD133 ~ + cells were isolated from human glioma tissue samples and subcultured in serum-free medium in vitro. The cells were suspended in suspension, then the CD133 immunomagnetic beads were used to isolate the positive cells in serum-free medium Under training. Nicardipine and mitoxantrone, respectively, or in combination on the above cultured cells were observed cell morphology, inhibition of proliferation and apoptosis rate changes. Results The cytotoxicity of nicardipine and mitoxantrone combined with CD133 immunomagnetic beads in the screened cells was observed by phase contrast microscopy. Some of the spheroids had disintegrated and the inhibition of proliferation was obvious. In the anthraquinone concentration-dependent manner, when mitoxantrone was administered in combination with nicardipine at 2.5 or 5.0 μmol·L -1, the dose of mitoxantrone at 10 -6 to 10 μmol·L -1 The inhibition of tumor cell proliferation in a concentration-dependent manner was significantly different from that of blank control and nicardipine (P <0.01), but not of CD133 immunomagnetic beads. Flow cytometry showed that nicardipine can cooperate with mitoxantrone to promote the apoptosis of test cells. Conclusion The high expression of ABCG2 in brain tumor stem cells is one of the reasons for its resistance to chemotherapeutic drugs. Nicardipine enhances the sensitivity of chemotherapeutic drugs through competitive inhibition of ABCG2.