人脑胶质母细胞瘤肿瘤干细胞的分离鉴定研究

Isolation and identification of tumor stem cells from human brain glioblastoma

目的 从人脑胶质母细胞瘤中分离出肿瘤干细胞.体外培养并鉴定其生物学特性.方法 获取5例人脑胶质母细胞瘤肿瘤细胞,通过无血清培养基和悬浮培养法,观察肿瘤干细胞克隆球的形成.利用免疫荧光技术检测原代肿瘤细胞和克隆球细胞中CD133阳性细胞的比例,分析克隆球细胞的抗原表达和多向分化能力.观察肿瘤干细胞子代分化细胞在无血清培养基中的逆向分化现象.结果 成功获取人脑胶质母细胞瘤肿瘤细胞,研究发现部分细胞具有克隆形成能力,在无血清培养基中呈悬浮球状生长,并可稳定增殖、传代.免疫荧光检测显示原代肿瘤细胞及克隆球细胞中均有CD133阳性细胞.克隆球细胞在含血清培养基中具有多向分化能力,其分化的子代细胞在无血清培养基中可逆向分化.结论 培养获得人脑胶质母细胞瘤肿瘤干细胞,证实其在体外具有自我更新、无限增殖、多向分化和逆向分化的生物学特性,为下一步研究奠定基础.

Objective To isolate tumor stem cells from human glioblastoma, culture and identity theer biOlOgiCal characteristics in vitro. Methods We obtained the tumor cells from 5 specimens of human glioblastoma, observed the formation of tumor stem cell spheres in simplified serum free medium by nanosphere suspension culture method. Immunoflorescence assay was used to determine the percentage of CD133 positive cells in primary tumor cells and floating tumor spheres and the expression of differentiations markers and multi-directional differentiation capacity of the tumor stem cell spheres were analyzed. The offspring cells of tumor stem cells were cultured in serum-free medium for observing their retrodifferentiation. Results Human primary glioblastoma cells were obtained successfully, part of them were capable of forming tumor suspension spheres in serum-free medium and able to proliferate and passage stably in vitro. Immunoflorescence assay shewed the existence of CD133 positive cells both in primary giiobiastoma cells and tumor spheres. The tumor stem cell spheres were differentiated multi-directionally in serum-containing medium. The offspring cells cultured in serum-free medium were able to re-proliferate and renew to form tu- mor stem cells. Conclusion The tumor stem cells isolated from human glioblastoma have potency of self-renewal, proliferation, multi-directional differentiation and retrodifferentiation in vitro.