人微血管内皮细胞通过分泌神经生长因子促进背根神经节细胞增殖机制研究  

Human vascular endothelial cell promoted the proliferation of dorsal root ganglion cells by the secretion of NGF

作　　者：原泉[1] 孙立[2] 陈建华[1] 安春厚[1] 

机构地区：[1]中国医科大学附属盛京医院骨外科,辽宁沈阳110003 [2]中国医科大学附属第一医院肾内科,辽宁沈阳110001

出　　处：《临床军医杂志》2016年第4期361-364,共4页Clinical Journal of Medical Officers

基　　金：辽宁省社会发展攻关计划(201225094);辽宁省高等学校优秀人才支持计划(LJQ2013086);沈阳市科技计划项目(F15-199-1-51)

摘　　要：目的探讨神经生长因子(NGF)在人微血管内皮细胞(HMVEC)促进背根神经节细胞(DRGC)增殖中的作用及分子机制。方法分离培养小鼠DRGC细胞,通过Transwell共培养小室与HMVEC进行非接触共培养,实验设HMVEC单独培养组、DRGC单独培养组、HMVEC与DRGC共培养组及PD90780组。PD90780组HMVEC使用NGF拮抗剂PD90780预处理1 h后,再与DRGC共培养。ELISA检测细胞上清液中NGF的水平;倒置显微镜观察各组DRGC生长情况;MTT检测细胞增殖水平;Real-time PCR检测GAP-43 mRNA水平;免疫荧光检测GAP-43水平;Western blot检测各组DRGC细胞中Ki-67、PCNA、GAP-43、ERK、p-ERK、AKT、p-AKT的表达。结果 HMVEC与DRGC共培养明显上调NGF水平。与DRGC单独培养组比较,HMVEC与DRGC共培养显著提高DRGC细胞增殖水平及增殖相关蛋白Ki-67、PCNA的表达,GAP-43 mRNA及蛋白水平均显著上调,ERK、AKT信号通路活化明显增加;与共培养组比较,PD90780处理组DRGC细胞增殖水平明显下降,Ki-67、PCNA、GAP43表达水平下调,ERK及AKT信号通路活化受到抑制。结论 HMVEC通过分泌NGF发挥促进DRGC增殖的作用,NGF拮抗剂能够逆转HMVEC对DRGC细胞增殖的影响。Objective To investigate the function and mechanism of nerve growth factor（ NGF） on the effect of human microvascular endothelial cells（HMVECs）promoting the proliferation in DRGCs. Methods Separated mouse dorsal root ganglion cells（DRGCs）, then using the Transwell chamber to establish the non-contact co-culture system with HMVECs. Four groups were created：DRGCs sin-gle culture group,HMVECs single culture group,co-culture of HMVECs and DRGCs group and PD90780 group. HMVECs were pre-treated with PD90780 for 1 hour in PD90780 group. The level of NGF in cell supernatant was detected by ELISA kits. Cell growth was observed under an inverted microscope. Cell proliferation of DRGCs was measured by MMT assay. The expression of GAP-43 was de-tected by Real-time PCR,immunofluorescence. The expression of Ki-67,PCNA,GAP-43,ERK,p-ERK,AKT,p-AKT was examined by Western blot. Results The level of NGF was up-regulated by the co-culture of HMVECs and DRGCs. Compared with DRGCs single culture guoup,the DRGCs proliferation rate was obviously increased in co-culture group,the expression of Ki-67,PCNA,GAP-43 and the activation of ERK,AKT pathway was significantly increased（P〈0. 05）. Compared with the co-culture group,PD90780 treatment decreased DRGCs proliferation rate,and the expression of Ki-67,PCNA,GAP43 obviously reduced. The activation of ERK and AKT signal pathway decreased as well. Conclusion HMVECs promoted the proliferation of DRGCs by the secretion of NGF,the NGF an-tagonist could reverse the influence of HMVECs on DRGCs proliferation.

关 键 词：人微血管内皮细胞 背根神经节细胞 共培养 神经生长因子 

分 类 号：R741[医药卫生—神经病学与精神病学]