胶质母细胞瘤肿瘤干细胞的分离培养与生物学特性研究

Isolation,culture and biological characteristics of tumor stem cells in human brain glioblastoma

目的从人脑胶质母细胞瘤中分离培养脑肿瘤干细胞(brain tumor stem cells,BTSC),并研究其生物学特性。方法利用无血清培养基和悬浮培养方法,从6例人胶质母细胞瘤标本中分离培养BTSC,并通过单克隆形成实验观察脑肿瘤干细胞球(brain tumor sphere,BTS)的形成过程。将BTSC接种于含血清培养基,观察其在体外的分化特点。将BTSC子代分化细胞更换培养条件,观察其在无血清培养基中的逆向分化现象。应用细胞免疫荧光染色对BTSC及其分化细胞进行鉴定。结果在人胶质母细胞瘤中成功分离出BTSC,其在无血清培养基中呈悬浮球状生长,具有很强的自我更新和增殖能力;免疫荧光显示其表达CD133。BTSC在含血清培养基中发生贴壁分化,分化后的子代细胞可表达CD133、神经元特异性烯醇化酶(NSE)和胶质纤维酸性蛋白(GFAP)。BTSC子代分化细胞在无血清条件下培养,能够再次增殖形成BTS,呈逆向分化现象。结论人胶质母细胞瘤中存在BTSC,其具有自我更新、无限增殖、多向分化以及逆向分化等生物学特性。

Objective To isolate brain tumor stem cells （BTSCs） from human glioblastoma and study their biological characteristics in vitro. Methods BTSCs were isolated from 6 fresh human glioblastoma specimens and cultured in simplified serum-free medium by nanosphere suspension culture method. The monoclonal formation experiment was performed to observe the formation of brain tumor stem cell spheres. BTSCs were inoculated onto serum-containing medium for observing their differentiation characteristics and then their offspring cells were cultured in the simplified serum-free medium for observing their retrodifferentiation. The immunofluorescence staining of cells was employed to identify the BTSCs and differentiated cells. Results BTSCs were isolated successfully from human glioblastoma, which presented as spheroid and suspension growths in simplified serum-flee medium, and had strong self-renewal and proliferation capacities with the expression of CD133, and as adhesion differentiation in the serum-containing medium, and the differentiated cells showed expressions of CD133, neurone-specific enolase （NSE） and glial fibrillary acidic protein （GFAP）. The progeny of BTSCs cultured in simplified serum-free medium could re-proliferate and form BTS, presenting as retrodifferentiation. Conclusion BTSCs existing in human gliohlastoma have biological characteristics of self-renewal, indefinite proliferation, pluripotent differentiation and retrodifferentiation.