转基因脑肿瘤小鼠模型中成体神经干细胞与脑肿瘤干细胞的克隆与鉴定

Isolation and identification of brain cancer stem cells and adult neural stem cells in genetically engineered brain cancer mouse models

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 mouse brain cancer stem ceils and adult neural stem cells from Tet-on genetically engineered brain cancer mouse models. Methods The cells from tumor tissue and SVZ of Tet-on genetically engineered brain cancer mouse models were cultured in DMEM/F12 mediume containing fibroblast grwoth factor-2 （hFGF）, epidermal growth factor （EGF） and B27 minus retinyl acetate. Differentiated cells of both cell spheres were cultured in DMEM/F12 containing 10% fetal calf serum（FCS）. The morphous and ultras tructures of both cell spheres were obeserved under phase contrast microscope and transmission electron microscope （TEM）. To determine whether both cell spheres contain side population （SP） cells, Hoechst 33342 was used to sort. Cell cycle and chromosome ploidy of both spheres cells were performed on flow cytometry. Multiple immunofluorescence was used to assay the ceil markers of both cell spheres and differentiated cells. Results Brain tumors were located at pineal region. The tumors were diagnosed as medulioblastoma. Cells from cancer tissue and SVZ formed stem-like sphere when cultured in Stem cell mediume and differentiated into neuronal-and glial-like cells when cultured in DMEM/F12 containing 10% fetal calf serum（FCS）. The uhrastructures of brain tumor sphere indicated stem cells characteristics of undevelopped organelles,high nuclear-cytoplasmic ratio. Heteroploid in mouse brain tumor spheres was 26.77%. All cells were diploid in neural stem cell sphere. A majority of cells in both spheres was nestin positive and small parts of the ceils were neuron-specific enolase （NSE） and glial fibrillary acidic protein （GFAP） positive. Most differentiated cells coexpressed nestin with MAP2 or GFAP. Conclusion Tet-on genetically engineered mouse brain cancer mouse model contains brain cancer stem cells and adult neural stem cells which have the capacity to self-renew and multidifferentiation.