血管内皮生长因子、碱性成纤维细胞生长因子-2及雌激素受体在早产儿视网膜病发病机制中的作用

The role of vascular endothelial growth factor, fibroblast growth factor-2 and estrogen receptor in the pathogenesis of retinopathy of neonatal mice

目的 测定新生小鼠视网膜正常发育及视网膜病时血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子-2（FGF-2）和雌激素受体（ER）表达的改变,了解它们在早产儿视网膜病（ROP）发病机制中的作用.方法 选择7日龄（P7）C57BL/6J新生小鼠474只,雌雄各半,分为吸氧组和对照组两大组,再按性别分成4小组,用逆转录聚合酶链式反应（RT-PCR）测定VEGF、FGF-2和ER的mRNA水平,用免疫组化测定它们的蛋白水平.具体分组及取材时间为：组1（n=126）,吸氧组,雌性;组2（n=126）,吸氧组,雄性,两组均自吸氧后每天分离视网膜至P21（P8～P21）行RT-PCR,每天摘眼石蜡包埋后行免疫组化;组3（n=111）,对照组,雌性;组4（n=111）,对照组,雄性,均自P7起每天分离视网膜至P17（P7～P17）行RT-PCR,自P7起每天摘眼至P21（P7～P21）行免疫组化.结果 （1）VEGF mRNA在正常小鼠自P7起上升,P9达高峰,P11起下降并始终维持低水平;在吸氧组自P8起显著下降,且在整个吸氧阶段持续降低,至出氧箱后缓慢上升,自P15起显著上升,并和对照组形成显著差异,受氧浓度的调节非常明显.FGF-2 mRNA在正常小鼠始终维持相对稳定的低水平;在吸氧组,吸氧期间无明显变化,出氧箱后3 d开始上升.ER mRNA在正常小鼠自P7起上升,P9达高峰,P11起下降并始终维持低水平;在吸氧组,吸氧期间变化与对照组相似,出氧箱后5 d开始上升,并维持较高水平直至P21.这3个细胞因子的蛋白水平变化均晚于其基因水平变化,但变化趋势基本相同.（2）性别和吸氧都不能单独影响上述细胞因子的表达（P＞0.05）;日龄可独立地影响它们的表达;吸氧和日龄两因素结合起来可显著影响它们的表达（P＜0.000 1）.结论 引起ROP发病的根本原因是早产,吸氧是其重要的外因;VEGF、FGF-2和ER参与血管的形成,在视网膜血管正常发育和ROP的发病机制中起重要作用;VEGF直接受氧浓度调节,在众多细胞因子中可能起关键性作用.

Purpose To investigate the changes of VEGF,FGF-2 and ER mRNA and protein levels in mice retinae of normal retinal vascular development and retinopathy, in order to elucidate risk factors and pathogenesis of retinopathy of prematurity（ROP）. Methods Four hundred and seventy-four 7-day-old（P7） C57BL/6J mice, half female and half male, were assigned to four groups according to hyperoxia and gender. Total RNA was extracted from 2 mice 4 retinae for one sample. VEGF, FGF-2 and ER mRNA expressions were determined by reverse transcription-polymerase chain reaction（RTPCR）. Their protein levels were determined by immunohistochemistry（IHC）. The groups were： female mice in group 1（n= 126） and male mice in group 2（n= 126） were exposed to 75% 02 for 5 days and returned to room air for another 5 days. Investigation was done everyday since exposure to hyperoxia until P17 for RT-PCR and P21 for IHC. Female mice in group 3（n = 111） and male mice in group 4（n= 111） were exposed to room air. RT-PCR was done everyday during P7 - P17, IHC was done everyday during P7 - P21. Results （1）The level of VEGF mRNA increased since P7, peaked at P9, and declined since Pll to a low level and maintained to P17 in normoxic groups. In hyperoxic groups, it declined since P8 and remained declining during oxygen exposure; while it rose slowly since mice were brought back to room air and rose rapidly since P15 which was significant compared to the controls, indicating a close relationship with changes of 02 concentration. The level of FGF-2 mRNA maintained low in normoxia; while in hyperoxic groups it had no changes during exposure to hyperoxia and rose since 3 days after back to room air and maintained to P21. The level of ER mRNA increased since P7, peaked at P9, declined since Pll and maintained to P17 in nomoxic group. In hyperoxic groups, it had no changes during the hyperoxic period and rose since 5 days after back to room air and maintained to P21. Changes of protein levels of these three factors were later than those of mRNA levels, but had the same trend. （2）Gender and hyperoxia didn＇t independently affect the expressions of VEGF, FGF-2 and ER mRNA（P〉0. 05）. However, the age was the independent factor which could affect their mRNA expressions（P〈0. 05）. While hyperoxia and age were integrated, these factors＇ mRNA expressions could be obviously affected（P〈0. 000 1）. Conclusions Our study suggested that hyperoxia followed by hypoxia was very important in the pathogenesis of ROP. VEGF, FGF-2, and ER played important roles on the development of normal retina vascularization and the pathogenesis of ROP. VEGF could be adjusted straightly by 02 concertration which might be the key factor in the pathogenesis of ROP.