血清剥夺法分离U87细胞系中的胶质瘤干细胞

背景：细胞周期分析已证实，90%以上的干细胞处于G0静止状态，因此采用不含任何生长因子和其他相关营养添加剂的血清剥夺培养基更适合筛选分离肿瘤干细胞。 目的：应用血清剥夺法培养胶质瘤U87细胞，并筛选鉴定该细胞系中的胶质瘤干细胞。 方法：将U87细胞培养在只含有DMEM和L-谷氨酰胺的培养基中培养6 d，筛选出胶质瘤干细胞，接着更换为神经干细胞培养基，观察细胞肿瘤球的形成过程；将肿瘤球接种于血清培养基，观察其在体外的分化特点；免疫荧光鉴定血清剥夺后尚存的细胞、增殖形成的肿瘤球细胞和分化细胞。 结果与结论：应用血清剥夺的方法成功地筛选出了表达CD133的肿瘤干细胞，并能增殖形成肿瘤球；肿瘤球可多向分化，子代细胞表达胶质纤维酸性蛋白和神经元特异性烯醇化酶。说明U87细胞系中存在具有自我更新及多向分化能力的胶质瘤干细胞。

Isolation of glioma stem cells from human glioma cell lines U87 with serum deprivation method

BACKGROUND: Cell cycle has proved that about 90% stem cells were in resting state in the G0 phase; therefore, serum deprivation method which did not contain any growth factors and related nutrition additive was beneficial for separating glioma stem cells. OBJECTIVE: To explore the method of serum deprivation to culture glioma cell lines U87, and to isolate and identify the cancer stem cells which are in the U87 cell lines. METHODS: U87 cells were cultured in the medium containing both DMEM and L-glutamine for six days; cancer stem cells would be screened out; then the medium was changed for the neural stem cell culturing medium. The formation of tumor spheres and their differentiation characteristics were observed when they were inoculated onto serum-containing medium. The immunofluorescence staining of cells was employed to identify the surviving cells cultured in serum deprivation medium, tumor spheres and differentiated cells. RESULTS AND CONCLUSION: The serum deprivation method was used to select tumor stem cells successfully which expressed CD133 and could form tumor spheres. The tumor spheres had an ability of multi-differentiations, and the daughter cells expressed glial fibrillary acidic protein and neuron specific enolase. The U87 cell lines exist glioma stem cells which have the capacities of self-renewal and multi-differentiation.