U251胶质瘤细胞系中脑肿瘤干细胞的分离、培养及鉴定

目的本研究旨在从U251胶质瘤细胞系中分离、培养和鉴定脑肿瘤干细胞,观察其的生长特征,为脑肿瘤干细胞的进一步研究打下基础。方法应用简化的无血清培养基和悬浮培养法培养U251细胞,并将获得的脑肿瘤干细胞接种于含血清培养基中观察其分化;交替用含血清培基和无血清培基培养U251细胞,观察其生长特性;用免疫荧光法检测脑肿瘤干细胞中CD133和Nestin的表达。结果 U251细胞接种至无血清培养基后,部分细胞能够存活并增殖为悬浮生长细胞球,并具有很强的自我更新和增殖能力;这种悬浮生长的细胞转移到含血清培基中后可贴壁分化;U251细胞在含血清培基和无血清培基中交替培养时能够快速转换生长方式;脑肿瘤干细胞中CD133和Nestin表达阳性。结论 U251胶质瘤细胞系中含一定比例脑肿瘤干细胞,此种细胞可在体外分离,培养和诱导分化。脑肿瘤干细胞的发现可能将为脑肿瘤研究提供全新的思路。

Isolation,Culture and Identification of Brain Tumor Stem Cellswithin U251 Glioma Cell Line In Vitro

Objective This study was to isolate,culture and identificate brain tumor stem cells from U251 Gliome cell line in vitro and observe their growth pattern.To establish a fundation for further reseach of brain tumor stem cell.Methods U251cells were seeded in serum-free DMEM-F12 medium supplemented with B27,EGF and bFGF in 6-well plates,after U251 brain tumor spheres formed,they were dissociated and passaged in fresh medium periodically.U251 brain tumor spheres and the single cells of them were induced to differentiate in the medium with 10% FBS supplemented.U251cells cultivate in serum-supplemented medium and serum-free medium alternately.At last we performed immunocytochemistry of U251 brain tumor spheres for CD133 and Nestin.Results Some U251 glioma cell have the capacity to self-renew,proliferate and generate free-floating neurosphere-like brain tumor spheres in serum-free medium in vitro.U251 glioma spheres and the single cells of them can be induced to differentiate and adherent to the bottom of the culture plates.The growing patterns of U251 glioma cell line can be converted by changing the culture mediums.The brain tumor stem cell are CD133＋Nestin＋ cells.Conclusion The U251 glioma cell line contain brain tumor stem cells which can be isolated,proliferated and differentiated in vitro.The research of brain tumor stem cells may provide a new platform for brain tumor study.