无血清培养基体外分离培养人胶质瘤干细胞与鉴定

Isolation, culture and identification of human gliom stem cells in serum-free medium in vitro

背景：最近有研究者采用神经干细胞的无血清培养基从脑肿瘤组织中培养出肿瘤干细胞。目的：探讨利用无血清培养基从原发胶质母细胞瘤组织中分离培养胶质瘤干细胞的可行性。设计、时间及地点：细胞学体外观察,于2008-08在山东省青岛市脑科研究所完成。材料：胶质瘤组织来自青岛大学医学院附属医院神经外科手术切除标本,DMEM/F12培养基、B27为GIBCO公司产品,碱性成纤维细胞生长因子、表皮生长因子为Peprotech公司产品。方法：无菌条件下取肿瘤深部无坏死囊变、未电凝组织,剪切消化成单细胞悬液后,加入含碱性成纤维细胞生长因子、表皮生长因子、B27添加剂的无血清DMEM/F12培养基,体外分离培养获得胶质瘤干细胞。待培养孔中细胞团数量增多、培养液刚刚变色时,吸取含有细胞团的培养液进行传代。取第3代胶质瘤干细胞,加入巢蛋白和CD133抗体行免疫荧光检测;加入含体积分数为0.1胎牛血清的DMEM/F12培养基诱导5d,行胶质纤维酸性蛋白免疫荧光染色观察分化情况。主要观察指标：原代与传代培养的胶质瘤干细胞形态及生长情况,胶质瘤干细胞巢蛋白、CD133的表达及其分化。结果：原代培养7-10d可形成大小不一的细胞团,球形或近似球形,呈悬浮或半悬浮状态生长,细胞形态均一,折光性好;传代24h后次代细胞团形成,细胞的大小、形态与原代无明显差别,连续传代5次后细胞团增殖活跃。第3代胶质瘤干细胞呈巢蛋白和CD133阳性表达,加入胎牛血清后细胞球贴壁分化,呈胶质纤维酸性蛋白阳性。结论：人脑胶质瘤组织中存在胶质瘤干细胞,在体外无血清条件下可保持未分化的悬浮状态;加入血清后贴壁分化呈胶质瘤细胞样或神经元样,符合干细胞的自我繁殖和多向分化特征。

BACKGROUND： Recently, some researchers cultured stem cells in neural stem ceLl serum-free medium from brain tumor. OBJECTIVE： To explore the feasibility about isolating and culturing gliom stem cells in serum-free medium from primary glioblastoma. DESIGN, TIME AND SETTING： The cytology in vitro experiments were performed at the Brain Research Institute, Qingdao City, Shandong Province in August 2008. MATERIALS： The glioma tissues were removed during operation at the Department of Neurosurgery of Affiliated Hospital of Medical College, Qingdao University. DMEM/F12 medium and B27 （GIBICO）, basic fibroblast growth factor （bFGF） and epidemal growth factor （EGF） （Peprotech） were used in this study. METHODS： The tissue without necrosis and electric coagulation in deep part of the tumor was sterilely removed. The tissue formed single cell suspension after shearing and digesting, and then incubated in DMEM/F12 medium with bFGF, EGF and B27 into single cell suspension. Glioma stem cells were obtained in vitro. While the quantity of cell group was increased and the culture solution＇s color was changed, the cell group was taken suction to transfer of culture. The third passage of glioma stem cells were detected by immunoflurescence after adding Nestin and CD133. Cells were incubated in the DMEM/F12 medium with 0.1 volume fraction of fetal bovine serum in culture solution for 5 days. Cell differentiation was observed by glial fibrillary acidic protein immunofluorescence staining. MAIN OUTCOME MEASURES： The form and growth of primary culture and passage of glioma stem cells, the expression of Nestin and CD133 and the differentiation of glioma stem cells were measured. RESULTS： The cell group was found under primary culture of 7-10 days. The cell group could grow with suspension or semi-suspension. The stem sells were observed with uniform shape and good refraction. The secondary cell groups formed after transfer of culture 24 hours, and there was no obvious difference in cell size and form compared with primary culture. The cell groups had an active proliferation after five times of serial passage. The third passage of glioma stem cells expressed Nestin and CD133, and they could adhere and differentiate after adding fetal bovine serum. After differentiation, they expressed GFAP. CONCLUSION： Human glioma stem cells exist in human brain glioma, and keep undifferentiated suspension form under serum-free medium in vitro. These cells present a glioma cell appearance or neuron appearance after adherence and differentiation in medium with serum. The cells were accordance with the character of stem cells in seff-reproduction and multiple directional differentiation.