人脑胶质瘤干细胞初步研究

Preliminary studies on tumor stem cells derived from human brain glioma

目的 从人脑胶质瘤体外细胞系和胶质瘤组织中分离、鉴定肿瘤干细胞，为进一步研究其生物学特性奠定基础。方法 将人胶质瘤SHG44细胞和手术标本制成的单细胞，分别用含血清培养基（DMEM＋10％FBS）和无血清培养基（DMEM／F12，添加bFGF、LIF和EGF）培养。用CD133免疫磁珠筛选，流式细胞仪和免疫荧光共聚焦显微镜检测干细胞、祖细胞和分化细胞特异性标志物。结果 SHG44细胞培养1周，用CD133磁珠分离得到的CD133^＋细胞的比例：血清组为0．021％，无血清组为1．2％。流式细胞仪检测：（1）Hoeehst33342一细胞比例：血清组为1．5％，无血清组为16．4％；（2）nestin^＋细胞比例：血清组为7．2％，无血清组为51．05％；（3）免疫磁珠分离的CD133^＋细胞再用流式细胞仪测得的CD133^＋细胞为83．02％，CD133^＋细胞群中有3．32％的CD133^＋细胞。标本源肿瘤细胞在无血清条件下培养两天后CD133^＋磁珠分离CD133^＋细胞比例为4％。CD133^＋细胞在分化不同阶段共表达或分别表达祖细胞标志物nestin、神经元和胶质细胞特异性标志蛋白MAP2和GFAP。结论 在胶质瘤细胞系和胶质瘤组织中均存在CD133^＋的脑肿瘤干细胞，具有自我更新和多向分化潜能、在无血清培养下细胞球体中CD133^＋细胞仍占少数，而nestin^＋细胞占多数，可作为进一步研究脑肿瘤干细胞生物学特性的实验材料在相关领域中的应用。

Objective Tumor stem cells were isolated and identified from established human glioma cell lines and glioma tissues, for the purpose of further evaluation of their biological characteristics. Methods SHG-44 cells cultured in vitro for long term and fresh surgical specimen were prepared in single ceil suspensions firstly, then cultured in serum containing medium （ DMEM supplemented with 10% FBS） and in serum free medium （DMEM/F12 plus bFGF, LIF and EGF）. The specific cell surface markers of tumor stem cells, tumor progenitor cells and differentiated tumor cells were detected with CD133 magnetic cell sorting, flow cytometry and immunofluorescence antibody assay under confocal microscope. Results SHC-44 ceils were cultured under different conditioned medium for a week, CD133^＋ cells were screened with CD133 magnetic ceil sorting. CD133^＋ cells accounted for 0. 021% of total SHG44 ceils in serum group, while in serum free group, percentage of CD133^＋ cells increased to 1.2%. Flow cytometry assay showed that （ 1 ） 1.5% of SHG 44 cells were Hoechst 33342 positive in serum group and 16.4% in serum free group; （2） The percentage of nestin positive cells in serum group was 7.2%, and 51.05% in serum free group; （3） After CD133 magnetic ceil sorting, the percentage of CD133^＋ cells was 83.02% , and there were 3.32% of CD133^＋ cells in the CD133^＋ cell group after magnetic cell sorting. Tumor cells from fresh surgical specimen were cultured in serum free medium for two days, then CD133^＋ cells were screened with magnetic cell sorting, and the percentage of CD133^＋ cells was 4%. CD133^＋ cells could co-express or express progenitor ceil surface marker nestin, glial ceil specific marker GFAP, and MAP2, respectively, in different differentiation stages. Conclusions Cell spheres containing brain tumor stem cells, which had capacities of self renewal and multi directional differentiation potentials, can be obtained both from long term in vitro cultured glioma cell lines and fresh glioma tissues by CD133 magnetic cell sorting and cultured in serum free medium. CD133^＋ cells were the minority in the cell spheres, while nestin＋ cells were the majority. These findings may help to provide experimental materials for further studies on brain tumor stem cells.