磁珠分选U251细胞系中的CD133^＋细胞及其生物学特性

Biological characteristics of CD133^+ cells in U251 cell line sorted by magnetic-activated cell sorting

背景:脑肿瘤干细胞理论认为,脑肿瘤干细胞是脑肿瘤细胞中"种子"细胞,是脑肿瘤发生、浸润和复发的关键细胞。目的:观察人多形性胶质母细胞瘤U251细胞系中CD133+细胞的增殖、分化及体内致瘤性等生物学特性。方法:运用磁珠分选技术分选U251细胞系中的CD133+和CD133-细胞亚群;MTT法绘制两个亚群细胞的生长曲线;单克隆形成率实验检测2个亚群细胞的增殖能力;免疫荧光检测CD133+细胞亚群的多向分化能力;裸鼠移植实验检测2个亚群细胞在裸鼠体内致瘤性的差异。结果与结论:U251细胞系中只有约4.5%的CD133+细胞;分选后的CD133+细胞能增殖形成典型的脑肿瘤干细胞球,生长曲线显示CD133+细胞增殖能力明显强于CD133-细胞;单克隆形成率实验显示CD133+细胞能形成脑肿瘤干细胞球的细胞比率达到78.5%~92.4%,而CD133-细胞仅有0.8%~2.4%;CD133+细胞能分化为具有GFAP和NeuN成熟表型的肿瘤细胞;CD133+的致瘤率为71.42%,而CD133-细胞无致瘤性。提示U251细胞系中存在少量具有增殖、多向分化与体内致瘤能力的CD133+细胞,CD133+细胞是符合肿瘤干细胞定义的细胞亚群。

BACKGROUND：The theory of brain tumor stem cells reports that brain tumor stem cell is the seed cell among brain tumor cells and is the key cell to occurrence, infiltration and recurrence of brain tumor. OBJECTIVE：To investigate the biological characteristics of CD133＋ cells in U251. METHODS：CD133＋ and CD133- subpopulation cells in the human GBM U251 cell line were sorted by magnetic-activated cell sorting. The purification of CD133＋ subpopulation was performed by flow cytometry. MTT and monoclone forming rate assay were performed to investigate the capacity of self-renewal and clonogenic potential of the subpopulation cells. Immunofluorescence staining was performed to investigate the multidiferentiation of CD133＋ cells. The tumorigenic potential of the two subpopulation cells in vivo was investigated by xenografting in BALB/c nude mouse brain. RESULTS AND CONCLUSION：Only 4.5% cells were CD133＋ in the total population of U251 cell line. The CD133＋ cells exhibited powerful proliferation capacity. The single CD133＋ cell, rather than CD133- cells, could proliferate and form typical brain tumor spheres. Cell growth curve showed that CD133＋ cells proliferated remarkably faster than CD133- cells while cultured in serum-free medium. Monoclone forming rate of CD133＋ and CD133- cells was 78.5-92.4% and 0.8-2.4% respectively. Immunofluorescence staining showed that CD133＋ cells could be induced to differentiate into mature neurocyte cells. Xenograft assay showed that CD133＋ cells had tumorigenic potential in vivo, while CD133- had not. The U251 cell line contained a small proportion of CD133＋ cells, which had the capacity for proliferation and multi- differentiation, and tumorigenic potential in vivo. The subpopulation of CD133＋ cells was the brain tumor stem cell subpopulation in U251.