脑胶质瘤干细胞鉴定与增殖及耐药特性的实验研究

目的 从胶质瘤组织中分离和培养出肿瘤干细胞,并初步探讨其生长特性. 方法 收集脑胶质瘤手术标本并获取细胞,用含有表皮生长因子(EGF)、白血病细胞抑制因子(LIE)和碱性成纤维生长因子(bFGF)的无血清培养液原代培养,再经免疫磁珠分离得到CD133+细胞.并用免疫细胞化学技术检测CD133、NSE和GFAP在细胞中的表达以鉴定CD133+细胞,比较不同恶性级别胶质瘤组织的CD133+细胞生长情况,并用CCK8法比较CD133+和CD133-细胞对替尼泊苷(VM-26)的耐药性. 结果 从胶质瘤组织中成功分选获得CD133+细胞,这些细胞能自我更新,增殖,并分化成NSE+和GFAP+的细胞.恶性度高的胶质瘤组织中CD133+细胞生长速度明显比低级别中的CD133+细胞快,且CD133+细胞在含有VM-26培养基中的存活细胞数显著多于CD133-细胞(P＜0.05). 结论 胶质瘤组织中存在肿瘤干细胞,这类细胞具有很强的耐药性,高恶性度胶质瘤组织中的CD133+细胞具有更强的增殖能力.

Identification of brain tumor stem cells and research on their characters of proliferation and drug-resistance

Objective To isolate and culture brain tumor stem cells (BTSCs) from glioma tissues and explore the biological characteristics of BTSCs. Methods Different grade glioma tissues were obtained from 20 clinical cases. After tumors were dissociated, the sample was triturated into the single cells and then filtered. The primary glioma cells were collected and cultured in the DMEM/F12 medium containing epidermal growth factor (EGF), leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF), in order to promote the proliferation of BTSCs. CD133 + cells were separated by immunomagnetic bead method and identified by testing the expressions of CD133, NSE and GFAP using immunocytochemistry. CCK8 was employed to assay the proliferating situation of CD133+ cells in the different grade gliomas, and to compare the drug resistance between the CD133+ and CD133- cells in the medium containing VM-26. Results CD133+ cells were successfully separated from glioma tissues.CD133+ cells proliferated by self-renewal, then differentiated into NSE+ cells and GFAP+ ones respectively. CD133+ cells in the high grade gliomas showed the faster generation than the ones in the low grade gliomas. CD133+ cells survived more easily than the CD133- cells in the medium containing VM-26. Conclusions BTSCs exist in the glioma tissues, and possess the more tolerant to the VM-26.CD133+ cells in the high grade glioma can proliferate much more easily.