EPO通过上调CyclinD1表达促进胶质母细胞瘤的体外增殖  

Erythropoietin promotes the proliferation of glioblastoma cells through up-regulation of Cyclin D1 in vitro

作　　者：王文涛[1] 唐兆华[1] 霍钢[1] 李英[1] 王海全[1] 叶小琳 刘自力[1] 谭赢[1] 石爽[1] 

机构地区：[1]重庆医科大学附属第一医院神经外科,重庆400016

出　　处：《重庆医科大学学报》2016年第8期787-791,共5页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:81306409);国家临床重点专科经费建设资助项目(编号:财社〔2011〕170号);重庆市教委科学技术研究资助项目(编号:KJ1500219);重庆市渝中区科技计划资助项目(编号:20150108)

摘　　要：目的:研究促红细胞生成素(erythropoietin,EPO)对胶质母细胞瘤体外增殖的影响,并探讨其机制。方法:体外培养胶质母细胞瘤U87细胞株,实验分为对照组、EPO处理组及EPO+细胞周期素D1(Cyclin D1)拮抗剂组。对照组:用等体积PBS替代EPO;EPO处理组:在U87细胞的培养基中加入EPO(0.1 U/ml)处理7d;EPO+cyclin D1拮抗剂组:U87细胞的培养基中同时加入EPO(0.1 U/ml)及Cyclin D1拮抗剂(5μmol/L)共处理7 d。用Cell Counting Kit-8(CCK-8)法测细胞增殖情况,细胞倍增时间检测各组细胞增殖速度,RT-PCR、Western blot和细胞免疫荧光法(immunofluorescence,IF)测定细胞周期关键蛋白Cyclin D1的m RNA和蛋白表达变化,流式细胞仪检测各组细胞周期。结果:与对照组相比,EPO处理组中U87细胞生长速度明显增快(CCK8:第3天P=0.014,第5天P=0.029,倍增时间:P=0.012),细胞中Cyclin D1的m RNA及蛋白表达水平也明显升高,差异有统计学意义(m RNA:P=0.001,蛋白:P=0.002,荧光定量:P=0.005),流式细胞仪检测发现U87细胞的增殖指数也明显升高(P=0.000);与EPO处理组相比,EPO+cyclin D1拮抗剂组细胞Cyclin D1 m RNA及蛋白的表达水平明显降低(m RNA:P=0.009,蛋白:P=0.000,荧光定量:P=0.013),流式细胞仪检测细胞增殖指数则有显著下降(P=0.000),同时细胞增殖能力和速度较EPO处理组明显降低(CCK8:第3天P=0.000,第5天P=0.000,倍增时间:P=0.001)。结论:本研究发现EPO可显著促进胶质母细胞瘤的体外增殖,上调细胞周期关键蛋白Cyclin D1的表达,加快细胞增殖周期可能是其重要的分子机制。Objective：To explore the effect of recombinant human erythropoietin（rh EPO）on the proliferation of glioblastoma cells in vitro and its mechanism. Methods：U87 cells were cultured in vitro and divided into three groups：control group treated with equal value of PBS;EPO treatment group cultured with a dose of 0.1 U/ml EPO for seven days;EPO ＋ Cyclin D1 antagonist group treated with EPO（0.1 U/ml）and Cyclin D1 antagonist（5 μmol/L）for seven days. The proliferative ability was measured by cell counting Kit-8 assay and double time;Cyclin D1 expression was detected by RT-PCR,Western blot and cell immunofluorescence,respectively. The cell cycle was analyzed by flow cytometry. Results：EPO promoted the proliferation of U87 cells and the m RNA and protein levels of Cyclin D1. Cyclin D1 antagonist inhibited the m RNA and protein levels of Cyclin D1 induced by EPO;U87 cells were blockaded at G1 stage,and the proliferation of ability was significantly reduced. Conclusion：EPO may significantly promote the proliferation of U87 cells through upregulation the expression of Cyclin D1;its molecular mechanism may relate with accelerated cell proliferation cycle.

关 键 词：促红细胞生成素 胶质母细胞瘤 CYCLIND1 细胞周期 增殖 CyclinD1拮抗剂 

分 类 号：R739.41[医药卫生—肿瘤]