阻断神经生长因子信号部分抑制雌二醇对恒河猴视网膜血管内皮细胞的作用  

PARTIALY INHIBITION OF THE EFFECTS OF ESTRODIOL ON RETINAL ENDOTHELIAL CELL BY NGF SIGNALING BLOCKAGE

作　　者：胡宝洋[1] 刘坤[1] 万瑾[1] 龚宏华[1] 佘振珏[1] 肖虹蕾[1] 周国民[1] 

机构地区：[1]复旦大学上海医学院解剖学与组织胚胎学系

出　　处：《解剖学报》2004年第6期612-616,共5页Acta Anatomica Sinica

基　　金：国家自然科学基金资助 (3 0 2 70 699) ;上海市曙光计划资助

摘　　要：目的　研究神经生长因子 (NGF)信号系统在雌激素调控视网膜血管内皮细胞中的作用。　方法　选用恒河猴视网膜 脉络膜血管内皮细胞系RF 6A ,RT PCR检测雌激素受体 (estrogenreceptor,ER)、NGF及其受体在视网膜血管内皮细胞中的表达。分别观察雌二醇 (estradiol,E2 )、NGF及VEGF、EGF、BDNF等对血管内皮细胞活力(MTT法 )的作用。用流式细胞术 细胞周期法检测E2 、NCF及E2 与K2 5 2a共同处理组的凋亡率。NGF抗体和选择性TrkA拮抗剂K2 5 2a研究对上述作用的阻断效应。同样的分组进行细胞划痕修复实验。 96孔板AngioMatrix胶上观察E2 及NGF对RF 6A内皮细胞管腔形成能力的影响。　结果　RF 6A在细胞外基质胶上可形成管腔样结构。RT PCR检测到ER α、NGF及NGF受体TrkA的mRNA。 1nmol L～ 1 0 0nmol LE2 可以剂量依赖性的增强细胞活力及单层细胞的划痕修复能力 ,其增强作用可部分被NGF抗体及 1 0 0nmol L的K2 5 2a选择性阻断。凋亡率检测显示各组凋亡率相近。E2 可促进RF 6A形成管腔 ,但不能被NGF抗体、TrkA阻断。　结论　除VEGF外 ,E2 还可通过NGF信号系统对视网膜血管内皮细胞活力、划痕修复起促进作用。但结果提示 ,阻断NGF信号系统不影响E2 促RF 6A形成管腔的作用。Objective To study the estradiolNGF regulatory cascade in retinal endothelial cells. Methods Retinachoroids vascular endothelial cell line RF/6A from rhesus was cultured in M199 medium contain 20% FBS.mRNA of ER,NGF and its receptors,TrkA and P75 NTR were detected with RTPCR.The cells were incubated with NGF,VEGF,antibodies against NGF or VEGF,and K252a(100nmol/L),the specific inhibitor of TrkA,separately or in different combination.MTT based cell counting assay was used to study the viability of the cells.The apoptosis was evaluated by FACS,mass migration by wound healing assay,and tubogenesis by AngioMatrix assay. Results We amplified the specific fragments of cDNA of ER,NGF and NGF receptor TrkA using RTPCR.10?nmol/L1?μmol/L estradiol augments the proliferation and increases the viability of RF/6A in a dosage dependent manner.In the wound healing based migration assay,we found the similar alteration.This effects of estradiol was partially blocked by NGF neutralized antibody and K252a.Apoptosis rates were at the similar level among the groups.For the tubogenesis of RF/6A,we found no augmentation by NGF,and no blocked augmentation by estradiol.Conclusion NGF,first identified as the survival factor of nerve system,now also seemed to be an activator of retinal endothelial cell,is under the regulation of estrogen.The proliferation and migration,but not the tubogenesis of retinal endothelial cells are regulated by this regulatory cascade.

关 键 词：雌激素 神经生长因子 视网膜 血管内皮细胞 管腔形成 恒河猴 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]