人脑胶质瘤干细胞增殖和分化过程中NOTCH-1信号通路的调控

背景：研究发现NOTCH-1信号通路在神经干细胞或神经前体细胞的自我更新、增殖及分化中起重要作用。 目的：探讨NOTCH-1信号通路在人脑胶质瘤干细胞增殖和分化过程中的调控作用。 设计、时间及地点：开放性实验，于2005-01/2007-03在解放军第三军医大学新桥医院完成。 材料：人脑胶质瘤组织、正常成人脑组织由解放军第三军医大学新桥医院神经外科提供。U251胶质瘤细胞株由解放军第三军医大学新桥医院神经外科吕胜青副教授惠赠；CHG-5胶质瘤细胞株由解放军第三军医大学西南医院病理研究所卞修武教授、姚晓红博士惠赠。 方法：取人脑胶质瘤组织、正常成人脑组织、U251及CHG-5胶质瘤细胞株，采用免疫磁珠法分选获得CD133+脑胶质瘤干细胞，加入DMEM/F12无血清培养基进行增殖培养，形成细胞克隆后，加入含体积分数为10%胎牛血清的培养液，2 h后行抗CD133和抗巢蛋白免疫荧光双标染色。 主要观察指标：人脑胶质瘤干细胞的生长和鉴定，采用WST-8法、免疫组化实验、流式细胞仪、免疫荧光双标实验检测NOTCH-1信号通路蛋白的表达。 结果：在无血清培养基中，细胞呈悬浮生长，培养24~48 h可见单个细胞开始分裂生长，形成肿瘤球，将肿瘤球转入含胎牛血清的培养基后，4 h周边细胞伸出突起并逐渐分化，24 h后肿瘤球迁移出的细胞增多，形成单细胞层。肿瘤球能同时表达干细胞标志物CD133和巢蛋白，CD133+脑胶质瘤干细胞核浆比例达2/3~3/4，突起少，胞浆中线粒体等细胞器较少，核糖体丰富，未见胶质丝等分化结构，符合干细胞超微结构特点。NOTCH-1蛋白在人脑胶质瘤组织中的表达明显强于正常成人脑组织（P < 0.01），在CD133+ U251及CHG-5胶质瘤细胞中有很强的表达，在GFAP+和MAP2+ U251及CHG-5胶质瘤细胞中的表达强弱不等，在MBP+ U251及CHG-5胶质瘤细胞中呈弱表达或不表达。在脑胶质瘤干细胞增殖过程中能检测到较强的NOTCH-1信号通路关键基因NOTCH-1，CBF-1，HES-1 mRNA及NOTCH-1，HES-1蛋白表达；而在细胞分化时NOTCH-1信号通路关键基因NOTCH-1，CBF-1，HES-1 mRNA和HES-1蛋白表达逐渐减弱。 结论：NOTCH-1信号通路的关键蛋白分子NOTCH-1在人脑胶质瘤组织和胶质瘤细胞株中均有表达，而在正常成人脑组织中仅微弱表达。NOTCH-1信号通路关键基因NOTCH-1和HES-1的表达强弱可能参与了胶质瘤干细胞增殖和分化的调控。

Regulatory effects of NOTCH-1 signaling pathway on the proliferation and differentiation of human brain glioma stem cells

BACKGROUND: NOTCH-1 signal pathway plays an important role in self-renewal, proliferation and differentiation of neural stem cells or neural precursor cells. OBJECTIVE: To analyze the regulatory effects of NOTCH-1 signaling pathway on the proliferation and differentiation of human brain glioma stem cells. DESIGN, TIME AND SETTING: The opening study was performed at the Xinqiao Hospital, Third Military Medical University of Chinese PLA from January 2005 to March 2007. MATERIALS: Human brain glioma tissue and normal adult brain tissue were obtained from the Department of Neurosurgery, Xinqiao Hospital, Third Military Medical University of Chinese PLA. U251 glioma cells were gifted by Associate professor Lü Sheng-qing from the Department of Neurosurgery, Xinqiao Hospital, Third Military Medical University of Chinese PLA. CHG-5 glioma cells were presented by Professor Bian Xiu-wu and Doctor Yao Xiao-hong from the Institute of Pathology, Southwest Hospital, Third Military Medical University of Chinese PLA. METHODS: Human brain glioma tissue, normal adult brain tissue, U251 and CHG-5 glioma cells were used to harvest CD133+ brain glioma stem cells by the immunomagnetic beads. DMEM/F12 serum-free medium was added. Following cell clone formed, the medium containing 10% volume fraction fetal bovine serum was added for 2 hours. Double staining (anti-CD133 and anti-nestin) was performed. MAIN OUTCOME MEASURES: Growth and determination of human brain glioma stem cells; Expression of Notch-1 signaling pathway protein was detected using WST-8 method, immunohistochemistry, flow cytometry and immunofluorescence staining. RESULTS: In the serum-free medium, cells were suspended. At 24-48 hours, single cells began to split and formed tumor mass, which was removed in the medium including fetal bovine serum. At 4 hours, peripheral cells extended processes and gradually differentiated. At 24 hours, tumor mass gradually moved away from cells and formed the monolayer. Tumor mass could express CD133 and nestin. The karyoplasmic ratio of CD133+ brain glioma stem cells reached 2/3-3/4, and there were a few processes, a few organelles and a plenty of ribosome. These were accorded with the characteristics of stem cells. NOTCH-1 protein expression was significantly stronger in the human brain glioma tissue compared with normal adult brain tissues (P < 0.01). NOTCH-1 protein strongly expressed in the CD133+ U251 and CHG-5 glioma cells, but had various expression in GFAP+ and MAP2+ U251 and CHG-5 glioma cells, and weakly or did not express in the MBP+ U251 and CHG-5 glioma cels. During brain glioma stem cell proliferation, there were strongly expressed Notch-1 signaling pathway key genes NOTCH-1, CBF-1, HES-1 mRNA and NOTCH-1, HES-1 protein expression. During cell differentiation, there was weakly expressed NOTCH-1, CBF-1, HES-1 mRNA and HES-1 protein expression. CONCLUSION: NOTCH-1 has been detected in human brain glioma tissue and glioma cells, but weakly expresses in the normal adult brain tissue. Hotch-1 and Hes-1 may take part in the proliferation and differentiation of glioma stem cells.