人微血管内皮细胞促进背根神经节细胞增殖的机制  

Mechanism of human microvascular endothelial cells promoting dorsal root ganglion cell proliferation

作　　者：原泉[1] 程扬[2] 杨亚帆[1] 孙立[3] 

机构地区：[1]中国医科大学附属盛京医院骨科,沈阳110004 [2]沈阳市骨科医院 [3]中国医科大学附属第一医院肾内科

出　　处：《中华实验外科杂志》2015年第7期1547-1551,共5页Chinese Journal of Experimental Surgery

基　　金：辽宁省自然科学基金资助项目（201102252）;辽宁省社会发展攻关计划资助项目（201225094）;辽宁省高等学校优秀人才支持计划资助项目（supported by Program for Liaoning Excellent Talents in University,LNET）;沈阳市科技计划项目（F15-199-1-51）

摘　　要：目的 探讨共培养条件下人微血管内皮细胞（HMVEC）对背根神经节细胞（DRGC）增殖的调控机制.方法 分离培养小鼠DRGC,采用Transwell半透膜建立双层细胞共培养体系,实验设DRGC单独培养组、DRGC与HMVEC共培养组、SU5416组和二甲基亚砜（DMSO）组.SU5416组预先用血管内皮生长因子受体2（Flk-1）抑制剂SU5416处理HMVEC 6 h后再与DRGC共培养.相差显微镜观察各组DRGC生长情况;噻唑蓝（MTT）检测DRGC细胞增殖;酶联免疫吸附试验（ELISA）检测共培养细胞上清液中血管内皮生长因子（VEGF）、神经生长因子（NGF）的表达;Western blot检测磷酸化Flk-1（p-Flk-1）、增殖细胞核抗原（PCNA）、细胞周期素（Cyclin） D1细胞外信号调节激酶1/2（ERK1/2）、磷酸化ERK1/2（p-ERK1/2）的表达.结果 与DRGC单独培养组比较,HMVEC共培养组DRGC增殖率显著增加,在培养12h后最为明显,增加39.07％ （P ＜0.01）,VEGF、NGF、PCNA、Cyclin D1、p-Flk-1、p-ERK1/2表达量明显增加（P＜0.05）.DMSO组与共培养组差异无统计学意义（P＞0.05）.与DMSO组比较,SU5416组DRGC增殖率显著降低,于培养12 h后下降最明显,降低23.93％（P＜0.01）.VEGF、NGF、PCNA、Cyclin D1表达量减少,Flk-1与ERK1/2磷酸化水平降低,p-Flk-1和p-ERK1/2相对表达量分别降低50.69％和23.12％ （P ＜0.01）.结论 Flk-1抑制剂可阻断HMVEC共培养对DRGC增殖促进作用,HMVEC共培养可能通过VEGF/Flk-1途径激活ERK/丝裂原活化蛋白激酶（MAPK）信号通路,进而促进DRGC增殖.Objective To explore the mechanism of human microvascular endothelial cells （HMVECs） to regulate the proliferation of dorsal root ganglion cells （DRGCs） under co-culture in vitro.Methods DRGCs from mice were isolated and cultured.A co-culture systerm of HMVECs and DRGCs was established.Four groups were created：DRGCs single culture group,co-culture of HMVECs and DRGCs group,dimethylsurfoxide （DMSO） group and SU5416 group.HMVECs were pretreated with SU5416 for 6 h in SU5416 group.Cell proliferation of DRGCs was measured by methyl thiazol tetrazolium （MTT） assay.The expression of vascular endothelial growth factor （VEGF） and nerve growth factor （NGF） was detected by commercially available enzyme linked immunosorbent assay （ELISA） kits.The expression of phosphorylated vascular endothelial growth factor receptor 2 （p-Flk-1）,proliferating cell nuclear antigen （PCNA）,Cyclin D1,extracellular signal-regulated kinase 1/2 （ERK1/2）,and phosphorylated ERK1/2 （p-ERK1/2） was examined by Western blotting.Results DRGC proliferation rate was increased by 39.07％ in co-culture group as compared with DRGCs single culture group after culture for 12 h （P ＜0.01）,and the expression of VEGF,NGF,PCNA,Cyclin D1,p-Flk-1,and p-ERK1/2 was significantly increased （P ＜ 0.05）.There was no obvious difference between DMSO group and co-culture group.Co-culture-induced DRGCs proliferation rate was decreased 23.93％ by SU5416 after culture for 12 h （P ＜0.01）.The expression of VEGF,NGF,PCNA,Cyclin D1 and phosphorylation level of Flk-1 and ERK1/2 were obviously reduced in SU5416 group as compared with DMSO group,and levels of p-Flk-1 and p-ERK1/2 decreased by 50.69％ and 23.12％ respectively （P ＜ 0.01）.Conclusion HMVEC may regulate DRGC proliferation by VEGF/Flk-1 signaling network and activation of ERK/mitogen-activated protein kinase （MAPK） pathway under co-culture in vitro.

关 键 词：人微血管内皮细胞 背根神经节细胞 共培养 增殖 

分 类 号：R73-36[医药卫生—肿瘤]