摘要
目的探讨胶质瘤细胞在缺氧条件下分泌的外泌体对胶质瘤干细胞迁移的作用。方法超高速离心法提取缺氧和常氧条件下胶质瘤细胞分泌的外泌体,采用透射电子显微镜(TEM)和动态光散射(DLS)、Western blot检测等方法对外泌体进行鉴定;用DIO染料标记外泌体,荧光显微镜观察胶质瘤干细胞能否摄取外泌体。将PBS(空白组)、常氧(20%O2,常氧组)和缺氧(1%O2,缺氧组)条件下提取的外泌体与胶质瘤干细胞共培养,CCK-8法测定胶质瘤干细胞增殖情况,Transwell实验检测胶质瘤干细胞迁移能力变化,Western blot检测JAK/STAT3通路蛋白磷酸化水平的改变。结果在胶质瘤干细胞的胞质中检测到外泌体携带的荧光染料DIO。CCK-8结果显示:提取的外泌体以不同浓度(浓度梯度依次为1、5、10、20、40μg/mL)处理不同时间(12、24、48、72 h)后,常氧组和缺氧组胶质瘤干细胞的细胞活力差异无统计学意义。与空白组和常氧组比较,缺氧组提取外泌体共培养组的胶质瘤干细胞迁移能力显著提高(P<0.05),JAK/STAT3通路蛋白磷酸化水平也显著增加(P<0.05)。结论胶质瘤细胞在缺氧条件下分泌的外泌体部分通过提高JAK/STAT3通路磷酸化水平促进胶质瘤干细胞迁移。
ObjectiveTo investigate the effect of exosomes secreted by hypoxia-exposed glioma cells on the migration ability of glioma stem cells in vitro.MethodsExosomes were isolated from glioma cells with hypoxia exposure or in normoxic culture by ultracentrifugation and identified by transmission electron microscopy,dynamic light scattering and Western blotting.The exosomes were labeled with DIO to allow observation of their intake by glioma stem cells under fluorescent microscope.The glioma stem cells were co-cultured with the exosomes or PBS for 24,48,or 72 h,and CCK-8 assay and Transwell assay were performed to assess the changes in the proliferation and migration ability of the cells.The changes in the phosphorylation levels of the proteins in the JAK/STAT3 pathway were detected using Western blotting.ResultsFluorescence signals were detected in the glioma stem cells co-cultured with the DIO-labeled exosomes.The results of CCK-8 assay showed that the glioma stem cells showed no significant changes in their proliferative activity after co-culture with the exosomes from the glioma cells with or without hypoxic treatment.Co-culture with the exosomes from hypoxia-exposed glioma cells enhanced the migration ability of glioma stem cells,as compared with treatment with PBS or exosomes derived from normally cultured glioma cells(P<0.05).The exosomes from hypoxia-exposed glioma cells also significantly increased the phosphorylation levels of the proteins in the JAK/STAT3 pathway.ConclusionCo-culture with exosomes from hypoxia-exposed glioma cells can enhance the migration ability of glioma stem cells partly by increasing the phosphorylation levels of the JAK/STAT3 pathway proteins.
作者
程玥
叶秀峰
CHENG Yue;YE Xiufeng(Department of Pathology,College of Basic Medical Sciences,Chongqing Medical University,Chongqing,400016,China)
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2020年第4期392-400,共9页
Journal of Third Military Medical University
