Yu Shi-Cang, Yi Liang, Zhou Zhi-Hua, Yao Xiao-Hong, Ping Yi-Fang, Bian Xiu-Wu
Institute of Pathology, Southwest Hospital, Third Military Medical University, Chongqing, 400038, PR China.
Ai Zheng. 2007 Dec;26(12):1388-91.
BACKGROUND & OBJECTIVE: Tumor stem cells (TSCs) possess the ability of chemoresistance. This study was to isolate and identify TSCs from human glioma cell line U87.
After U87 cells were grown as monolayer attached at the bottom of flasks in the medium containing serum, neural stem cell culture medium containing 5 ng/ml vincristine was added to obtain the first generation of tumorosphere. Then, the first generation of tumorosphere was dissociated into single cell suspension and seeded in the neural stem cell culture medium to obtain the second generation of tumorosphere. The morphology of the first and the second generations of tumorosphere was observed by light and scanning electron microscopy, respectively. The expression of nestin, glial fibrillary acidic protein (GFAP), beta-tubulin III and myelin basic protein (MBP) in the second generation of tumorosphere and differentiated cells were determined by immunofluorescence assay and confocal laser scanning microscopy.
The first and second generations of tumorosphere were obtained successfully. Cells inside the second generation of tumorosphere with a few protrusions attached with each other tightly. The expression of nestin, a stem cell marker, was detected in the second generation of tumorosphere. The expression of nestin, GFAP, beta-tubulin III, MBP was detected in the differentiated cells derived from the second generation of tumorosphere.
Tumor stem cells with the capacity of self-renewal and multipotency exist in U87 cells. Selective culture of tumor cells with treatment of vincristine might be a simple and practical method for isolation of TSCs.
肿瘤干细胞（TSCs）具有化疗耐药能力。本研究旨在从人胶质瘤细胞系U87中分离并鉴定肿瘤干细胞。
将U87细胞在含血清的培养基中贴壁生长于培养瓶底部后，加入含5 ng/ml长春新碱的神经干细胞培养基以获得第一代肿瘤球。然后，将第一代肿瘤球解离成单细胞悬液，并接种于神经干细胞培养基中以获得第二代肿瘤球。分别通过光学显微镜和扫描电子显微镜观察第一代和第二代肿瘤球的形态。通过免疫荧光分析和共聚焦激光扫描显微镜检测第二代肿瘤球及其分化细胞中巢蛋白、胶质纤维酸性蛋白（GFAP）、β-微管蛋白III和髓鞘碱性蛋白（MBP）的表达。
成功获得第一代和第二代肿瘤球。第二代肿瘤球内的细胞有一些突起，彼此紧密相连。在第二代肿瘤球中检测到干细胞标志物巢蛋白的表达。在源自第二代肿瘤球的分化细胞中检测到巢蛋白、GFAP、β-微管蛋白III、MBP的表达。
U87细胞中存在具有自我更新和多能性的肿瘤干细胞。用长春新碱处理选择性培养肿瘤细胞可能是一种简单实用的分离肿瘤干细胞的方法。
