Tet-off/Tet-on基因工程鼠中脑肿瘤干细胞的克隆与鉴定*

目的:从Tet-off/Tet-on 转基因脑肿瘤小鼠模型中分离鉴定脑肿瘤干细胞，为进一步研究其是否起源于神经干细胞奠定基础。方法:从出现临床症状的转基因小鼠天幕区肉眼可见的肿瘤组织中取材，一部分做冰冻切片；另一部分制成单细胞悬液，培养于含生长因子的无血清培养基和含血清培养基。在光镜下观察常规病理，用相差显微镜和电镜分别观察细胞形态及其超微结构，用流式细胞仪检测Hoechst33342 阴性的SP细胞，以及其染色体倍体和细胞周期，免疫荧光染色检测细胞球体及其分化细胞中的神经巢蛋白（Nestin）、神经元特异性烯醇化酶（NSE）、glialfibrillaryacidicprotein（GFAP）表达情况。结果:肿瘤位置均位于小脑天幕上下约2～3mm的松果体区域内，常规病理诊断为髓母细胞瘤。肿瘤细胞在无血清培养条件下，呈悬浮的球状生长，在含血清培养条件下贴壁分化成瘤性胶质细胞、神经元。电镜见到了核质比高、细胞器不发达、具干细胞特征的细胞。流式细胞仪检测肿瘤细胞球中的SP（sidepopulation）细胞为17.27% ，异倍体细胞为26.77% 。免疫荧光染色发现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 。与之相反，贴壁细胞大部分表达分化标志物微管相关蛋白2（MAP2）或胶质纤维酸性蛋白（GFAP），同时共表达干细胞标志物Nestin。结论:Tet-off/Tet-on 转基因小鼠脑肿瘤组织中存在具有自我更新和多向分化潜能的脑肿瘤干细胞。 

Isolation and Characteristics of Brain Tumor Stem Cells in Genetically Engineered Mouse Models

Objective:To isolate and identify mouse brain tumor stem cells from Tet-off/Tet-on genetically engineered mouse models with brain tumors, and to explore the origin of brain tumor stem cells. Methods: Brain tumor tissues were obtained from genetically engineered mice with brain tumor symptoms. Cells from the tumor tissue were cultured in DMEM/F 12medium containing fibroblast growth factor-2 (bFGF), epidermal growth factor (EGF) and B27minus retinyl acetate. Differentiated cells of mouse brain tumor spheres were cultured in DMEM/F 12containing 10% FCS. Cell morphology and ultrastructure were observed using phase contrast microscopy and transmission electron microscopy (TEM). To determine whether mouse brain tumor spheres contained candidate cancer stem cells, Hoechst 33342 fluorescent staining was used to sort for the side population (SP) phenotype. Cell cycle stage and chromosome ploidy of mouse brain tumor sphere cells were detected using flow cytometry. Immunofluorescence was used to assay for markers of brain tumor spheres and differentiated cells. Results: Brain tumors were located in the pineal region. The tumors were di-agnosed as medulloblastoma. Cells from genetically engineered mouse brain tumor tissue formed stem-like spheres when cultured in DMEM/F 12medium containing FGF2, EGF and B 27minus retinyl acetate, and dif -ferentiated into neuronal and glial cells when cultured in DMEM/F 12containing 10% FCS. Using phase contrast and transmission electron microscopy, the brain tumor spheres showed stem cell ultrastructural characteristics including undeveloped organelles and a high nucleus:cytoplasm ratio. The percentage of SP cells in brain tumor spheres was 17.27% . Heteroploidy occurred in 26.77% of the mouse brain tumor spheres. The majority of cells in the mouse brain tumor spheres were Nestin positive and a small fraction of cells were NSE and GFAP positive. Most differentiated cells coexpressed Nestin with MAP 2 or GFAP. Conclusion:Tet-off/Tet-on genetically engineered mouse brain tumor contains brain tumor stem cells which have the capacity to self-renew and differentiate into neuronal and glial cells.