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目的从Tet-on转基因脑肿瘤小鼠模型中分离鉴定成体神经干细胞和脑肿瘤干细胞。方法分别从转基因小鼠肿瘤和室管膜下区取材,将细胞培养于含生长因子的无或含血清培养基。光镜下观察病理,相差显微镜和电镜下分别观察细胞形态及超微结构,流式细胞术检测Hoechst33342阴性的SP细胞、染色体倍体和细胞周期,免疫荧光染色检测细胞球体及分化细胞表面标志物。结果无血清培养条件下,肿瘤细胞和室管膜下区细胞均呈悬浮球状生长,含血清培养条件下贴壁分化。成体神经干细胞球细胞较小,形态相对较规则。电镜下两者均表现核质比高、细胞器不发达、具干细胞特征,但在细胞器发育上有所差异。肿瘤细胞球中异倍体细胞为26.77%,而神经球中细胞均为二倍体。免疫荧光染色两种细胞球中大部分细胞表达干细胞标志物Nestin,少数细胞表达分化标志物GFAP和NSE。贴壁后两者的大部分细胞表达分化标志物,且部分细胞共表达前体细胞标志。结论Tet-on转基因脑肿瘤小鼠中存在具有自我更新和多向分化潜能的脑肿瘤干细胞和神经干细胞。
Objective To isolate and identify adult neural stem cells and brain tumor stem cells from a mouse model of Tet-on transgenic brain tumor. Methods Tumor and subependymal zone of transgenic mice were harvested separately, and the cells were cultured in growth medium containing or without serum. The morphological and ultrastructural changes of Hoechst33342 cells were observed by light microscopy and phase contrast microscopy and electron microscopy. The Hoechst33342 negative SP cells, chromosome ploidy and cell cycle were detected by flow cytometry. The cell spheres and differentiated cell surface markers were detected by immunofluorescence staining . Results Under the condition of serum-free culture, the cells in the tumor cells and the subependymal cells all grew in a suspended globular form, and adhered to the cells under the condition of serum-containing culture. Adult neural stem cells are small, relatively regular morphology. Both electron microscopy showed high ratio of nuclear to cytoplasm, organelles underdeveloped, with stem cell characteristics, but there are differences in organellar development. The aneuploid cells in tumor cells were 26.77%, while the cells in neurospheres were diploid. Immunofluorescence Staining Most cells in both cell spheres expressed the stem cell marker Nestin, while a few cells expressed the differentiated markers GFAP and NSE. Most of the adherent cells express differentiated markers, and some cells co-express precursor cell markers. Conclusion There are brain tumor stem cells and neural stem cells in Tet-on transgenic brain tumor mice with self-renewal and multi-directional differentiation potential.