胶质瘤中肿瘤干细胞的分离、培养及鉴定  

Isolation, culture and identification of brain tumor stem cells within glicoma tissue in vitro

作　　者：邓永文[1] 方加胜[1] 李茗初[1] 陈风华[1] 刘劲芳[1] 伍军[1] 周向阳[1] 方芳[2] 卢明[1] 陈成[1] 周仁辉[1] 曾飞跃[1] 

机构地区：[1]中南大学湘雅医院神经外科,湖南长沙410008 [2]中南大学湘雅医院心血管内科,湖南长沙410008

出　　处：《中国现代医学杂志》2005年第16期2449-2452,共4页China Journal of Modern Medicine

基　　金：湖南省科技厅重点资助项目(NO.02JTY2009)

摘　　要：目的在胶质瘤中分离,培养及鉴定脑肿瘤干细胞,观察脑肿瘤干细胞的生长特性,建立脑肿瘤干细胞分离、培养及鉴定的方法,为脑肿瘤干细胞的进一步研究打下基础。方法术中取胶质瘤细胞,接种于含生长因子的无血清培养基中培养,使其中的脑肿瘤干细胞增殖,并进行有限稀释实验确定肿瘤比例及增值能力;利用细胞免疫荧光及组织免疫组化法检测脑肿瘤干细胞在细胞培养或组织切片中CD133和Nestin的表达。结果在胶质瘤中,只有大约1%的细胞能在无血清培养基中存活并悬浮生长,并增殖形成克隆性脑肿瘤干细胞球,传代后脑肿瘤干细胞仍保持很强的自我更新和增殖能力;体外培养中脑肿瘤干细胞表达神经干细胞的特异性标志物Nestin和CD133,在肿瘤组织切片中表达CD133阳性细胞。结论胶质瘤组织中存在极少数的脑肿瘤干细胞(大约为1%),并能在体外将其分离,培养;神经干细胞可能是脑肿瘤干细胞的起源细胞。[Objective] Isolation, culture and identification of brain tumor stem cells （BTSCs） within glicoma tissue in vitro. Observe the growth pattern of BTSCs, and to investigate their expression of specific markers. To establish a Isolation, culture and identification method for BTSCs. [Methods] Tumor from patients undergoing tumor resections were acutely dissociated, triturated into single cells in sterile DMEM-F12 medium and then filtered. The tumor cells were seeded at a concentration of 200 000 viable cells per mL into serum-free DMEM-F12 medium simply supplemented with B27 （1：50）, human basic fibroblast growth factor （20 μg/L）, human epidermal growth factor （20 μg/L）, insulin （4 u/L）, L-Glutamine, penicillin and streptomycin. After the primary brain tumor spheres （BTS） generated, they were triturated again and passaged in fresh medium. To determine the number of cells from pools population of primary tumor cells that possess sphere-forming capacity, limiting dilution assay was performed. At last we performed immunocytochemistry of BTSs for CD133 and Nestin expression and immunohistochemistry of tumor specimen sections for CD133^＋ cells. [Results] Only about 1% cells in tumors of glicoma tissue has the capacity to self-renew, proliferate and generate free-floating neurosphere-like BTSs in medium in vitro. The BTSCs are CD133^＋ Neatin^＋ cells. [Conclusion] Glicoma tissue contain CD133^＋Neatin^＋ tumor stern cells which can be isolated, prolifer- ate and differentiate in vitro and give rise to brain tumor spheres. Normal neural stem cell （NSCs） may be origin cells of BTSCs.

关 键 词：肿瘤干细胞 分离培养 鉴定 CD133 NESTIN 胶质瘤 

分 类 号：R73-351[医药卫生—肿瘤] R730.264[医药卫生—临床医学]