人脑胶质瘤中肿瘤干细胞的体外培养及生物学特性

背景：有研究者提出，脑内存在少量正常的神经干细胞能够向肿瘤组织迁移。这需要对从胶质瘤体外培养得到的肿瘤干细胞与正常的神经干细胞进行鉴别。目的：从人脑胶质瘤组织中分离脑胶质瘤干细胞进行体外培养，并对其干细胞特性加以鉴定，观察脑肿瘤干细胞的生长特性。设计、时间及地点：细胞学观察实验，于2007-02/12在解放军第四军医大学细胞工程研究中心完成。材料：7份肿瘤标本来源于胶质瘤患者，间变性星形细胞瘤标本3份，多形性胶质母细胞瘤标本4份。方法：将获取的胶质瘤细胞置于含2%B27、表皮细胞生长因子、碱性成纤维细胞生长因子、左旋谷胺酰氨、胰岛素、青霉素和链霉素生长因子的无血清DMEM/F12培养基中，重新悬浮为单细胞悬液，以1×108 L-1接种于含有B27、表皮细胞生长因子及碱性成纤维细胞生长因子的DMEM/F12培养基中培养分离培养肿瘤干细胞球。取第5代的肿瘤干细胞球，离心后除去原培养基，用含有体积分数为0.10胎牛血清的DMEM/F12培养基接种于放置有多聚赖氨酸包被盖玻片的小平皿中，观察肿瘤干细胞分化情况。主要观察指标：利用细胞免疫荧光及组织免疫组织化学法检测脑肿瘤干细胞在细胞培养或组织切片中CD133及巢蛋白表达。结果：在胶质瘤中存在一定量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，细胞核较大，核质比例高，具有肿瘤细胞的特性，与原肿瘤组织标本的苏木精-伊红染色比较，肿瘤干细胞分化后的子代细胞中大部分与胶质瘤细胞相似。原代及传代脑肿瘤干细胞表达神经干细胞的特异性标志物巢蛋白和CD133。结论：人脑胶质瘤中存在一定量的肿瘤干细胞，并能在体外将其分离培养，能够自我更新增殖、诱导分化，表达神经干细胞标志物CD133。

Culture and biological characteristics of tumor stem cells from human brain glioma tissue in vitro

BACKGROUND: Some studies proved that a few neural stem cells of brain could migrate to tumor tissues. The brain tumor stem cells (BTSCs) isolated from glioma tissue in vitro should be identified from neural stem cells. OBJECTIVE: To isolate and culture BTSCs from human glioma tissue in vitro, to identify the stem cell characteristics of BTSCs, and to investigate the growth of BTSCs. DESIGN, TIME AND SETTING: Cellar morphological observation was achieved in Cell Engineering Research Center, the Fourth Military Medical University of Chinese PLA from February to December in 2007.MATERIALS: Seven samples were selected from glioma patients, comprised of 3 anaplastic astrocytoma ones and 4 glioblastoma multiforme ones.METHODS: BTSCs were seeded into serum-free DMEM/F12 medium supplemented with 2% B27, human basic fibroblast growth factor, human epidermal growth factor, insulin, L-glutamine, penicillin and streptomycin. Single cell suspension at a concentration of 1×108/L cells were inoculated on DMEM/F12 medium supplemented with 2% B27, human basic fibroblast growth factor and human epidermal growth factor so as to isolate and culture brain tumor spheres. After the removal of primary medium, the brain tumor spheres at the fifth passage were centrifuged and seeded on the polylysine-coated cover slice using DMEM/F12 medium added with 0.10 volume fraction of fetal calf serum. The differentiation of BTSCs was observed.MAIN OUTCOME MEASURES: Immunocytochemistry and immunohistochemistry were performed to detect the CD133 and Nestin expression of BTSCs. RESULTS: A certain amount of cells in glioma tissue had the capacity to self-renew, proliferate and generate flee-floating neurosphere-like BTSCs in serum-free medium. Cell nucleus was relatively bigger, and nuclear-cytoplasmic ratio was high, which were all characteristics of tumor cells. Compared with hematoxylin-eosin stained samples in primary tumor tissues, daughter cells differentiated from BTSCs were partly identical with glioma cells. Both primary and passaged BTSCs expressed CD133 and Neatin, which were two specific markers of nerve stem cells. CONCLUSION: Tumor stem cells in human brain glioma tissue can be isolated and proliferated in vitro, and they can self-renew, differentiate and express CD133.