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目的从胶质瘤组织中培养、分离肿瘤干细胞,并初步探讨其生物学特性。方法采集8例人脑胶质瘤手术标本,剪碎、胰酶消化,滤网过滤收集细胞。淋巴细胞分离液除去其中的红细胞,用含EGF、LIF和bFGF的无血清培养液培养,形成细胞球体后经免疫磁珠分离获取CD133+细胞, 用有限稀释法继续在上述无血清培养基中培养得到肿瘤细胞球后连续传代培养。取第5代肿瘤干细胞,用含10％FBS培养液诱导分化。分化前后用Nestin、MAP2、GFAP免疫细胞化学染色检测肿瘤干细胞及其分化细胞的相应标志物。结果在1例间变性星形细胞、室管膜细胞混合瘤中克隆到了 CD133+细胞,这些细胞在体外培养时,能稳定维持于球形生长状态(3个月,14代),在适合的环境中随时能自我更新和增殖,并分化成MAP2+的瘤性神经元和GFAP+的瘤性胶质细胞,而CD133-细胞则无此特性。结论在胶质瘤组织中有肿瘤干细胞存在,这些细胞在体外能长期培养和传代,可为进一步研究胶质瘤的细胞生物学和分子生物学特征开拓新途径。
Objective To culture and isolate tumor stem cells from glioma tissue and to investigate its biological characteristics. Methods Eight human glioma specimens were collected, cut, digested with trypsin and filtered through a filter. Lymphocytes were separated from the erythrocytes and cultured in serum-free medium containing EGF, LIF and bFGF. After forming spheroids, CD133 + cells were isolated by immunomagnetic beads and cultured in the above serum-free medium by limiting dilution Tumor cell ball after continuous subculture. Generation 5 cancer stem cells were seeded in 10% FBS medium to induce differentiation. Before and after differentiation, Nestin, MAP2 and GFAP immunocytochemistry were used to detect the corresponding markers of tumor stem cells and their differentiated cells. Results CD133 + cells were cloned in a mixed tumor of astrocytes and ependymal cells. These cells could be maintained in spherical growth state (3 months and 14 passages) when cultured in vitro. In a suitable environment Can self-renew and proliferate at any time, and differentiate into MAP2 + neoplastic neurons and GFAP + neoplastic glial cells, while CD133- cells do not have this characteristic. Conclusion There are tumor stem cells in glioma tissues. These cells can be cultured and passaged in vitro for a long time, which may be a new way to further study the cell biology and molecular biology of glioma.