缺氧诱导终末胶质瘤细胞逆分化为胶质瘤干样细胞-第三军医大学学报.docVIP

关于本论文学术方面的意见： 修改说明：本研究旨在说明缺氧可以诱导分化期胶质瘤细胞发生逆分化形成肿瘤干样细胞。我们经过磁珠分选得到CD133-细胞群，然后对该细胞群进行缺氧培养。采用单细胞成球（缺氧成球能力明显强于常氧培养组），免疫荧光、RT-PCR、Western bloting检测了肿瘤干细胞标记蛋白（SOX-2、OCT-4、KLF-4、Nanog、CD133），结果显示缺氧组均高表达，并通过流式细胞仪证明缺氧培养后CD133+细胞数比例逐渐增高。为了验证此理论的正确性，我们还通过细胞周期及凋亡予以验证，结果表明，缺氧培养CD133-细胞群后细胞周期阻滞于G0/G1期，同时抗凋亡能力也明显强于常氧培养组，这些特性也符合了胶质瘤干细胞特性。因此，我们采用上述方法予以证明缺氧可以诱导胶质瘤细胞逆分化形成肿瘤干样细胞。本研究结果可以很好解释胶质瘤术后为何高复发，并为高压氧治疗胶质瘤提供另一科学依据。同时，本研究也证明了胶质瘤干细胞除了来源于正常神经干细胞突变外，胶质瘤细胞发生逆分化形成胶质瘤干样细胞也可能是其来源的重要途径之一。 缺氧诱导胶质瘤细胞肿瘤干样细胞形成体外初步研究 汪攀，赵秀文，兰川，吴南 （400038 重庆， 第三军医大学西南医院神经外科） [摘要]目的 探讨胶质瘤细胞方法 采用CD133+细胞免疫磁珠分选法分离得到CD133-细胞CD133- U87、GL261细胞系制成单细胞悬液并种植一个细胞于96孔板内，分别行缺氧及常氧培养，观察单细胞生长情况。（3）缺氧处理CD133-细胞48小时后行免疫荧光染色鉴定肿瘤干细胞样标记蛋白（SOX2，OCT-4，KLF-4，Nanog，CD133）表达情况，并分别缺氧处理03、6、9、12、24 h及012、24、48、72 h行PCR及检测CD133+细胞缺氧处理胶质瘤细胞结果 CD133-胶质瘤荧光 肿瘤干细胞样标记蛋白CD133-胶质瘤细胞缺氧处理48h后PCR及PCR显示SOX-2，OCT-4及Nanog最高表达在缺氧后9h，KLF-4及CD133在缺氧后12h表达最高OCT-4最高表达在缺氧后12h，CD133表达最高在缺氧KLF-4及 Nanog最高表达则在缺氧48h后。CD133+细胞结论 缺氧可以诱导CD133-胶质瘤细胞肿瘤干样细胞。 [关键词] 缺氧； 胶质瘤； 肿瘤干样细胞； Cancer stem-like cells can be induced under hypoxia conditions in glioma cells Wang Pan, Zhao Xiuwen, Lan Chuan, Wu Nan (Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing, 400038, China） [Abstract] Objective To explore stem-like cells can be induced under hypoxia conditions in glioma cells. Methods U87, GL261 and Primary glioma cells were cultured in DMEM/F12+10%FBS and CD133- cells were sorted through magnetic-activated cell sorting (MACS). Single CD133- cell was transferred to 96-well plates (n=6) containing 170μl serum free DMEM/F12 medium. Three of them were cultured in 1%O2, and others in 21%O2 for 21 days; at 0, 3, 7, 14 or 21 day the number of formed neurospheres was recorded. The markers of CSCs (CD133, SOX-2, OCT-4, KLF-4 and Nanog) were detected through immunofluorescence staining after cells exposed hypoxia 48h. qRT-PCR and Western bloting were used to detect the change of RNA and protein of cancer stem cell markers respectively un