rAAV2-NTF2对糖尿病大鼠血视网膜屏障的保护作用  

Protective effect of rAAV2-NTF2 on blood-retinal barrier damage in diabetic rats

作　　者：史煜[1,2] 李斌[1] 李涛[1] 曹亮[1] 李永浩[1] 马红婕[1] 谢琳[1] 唐仕波[1] 

机构地区：[1]中山大学中山眼科中心,眼科学国家重点实验室,广东广州510060 [2]南京医科大学附属南京第一医院眼科,江苏南京210006

出　　处：《中国病理生理杂志》2009年第4期759-763,共5页Chinese Journal of Pathophysiology

基　　金：“十五”国家科技攻关计划资助项目(No.2004BA702B);国家自然科学基金资助项目(No.30772392)

摘　　要：目的:观察2型重组腺相关病毒-核运输因子2(rAAV2-NTF2)对糖尿病大鼠血视网膜屏障功能损伤的拮抗作用。方法:伊凡思蓝(EB)灌注铺片观察糖尿病大鼠视网膜血管分布和形态。亚克隆构建pSNAV-NTF2载体,腺相关病毒包装形成rAAV2-NTF2。成年雄性Wistar大鼠,以右眼为实验眼,玻璃体腔注射4μLrAAV2-NTF2,左眼为对照眼,玻璃体腔注射rAAV2-增强型绿色荧光蛋白(EGFP)4μL。1个月后取4只大鼠左眼,固定后取视网膜制备全视网膜铺片,荧光显微镜下观察EGFP表达情况。再行STZ腹腔注射诱导糖尿病,同时设单纯糖尿病组(diabetes,D组)和正常组(normal,N组)。糖尿病1个月后以45 mg/kg剂量静脉注射EB,循环2 h后1%多聚甲醛枸橼酸缓冲液灌注并摘除眼球取视网膜,置于甲酰胺中浸泡提取染料,用分光光度计测量甲酰胺中染料的浓度。结果:正常组大鼠可见在低背景荧光下视网膜血管对EB有很好的屏障作用,糖尿病2周大鼠视网膜血管仅见背景荧光增强,糖尿病1月后血管出现异常节段性扩张,局部血管周围EB渗漏。糖尿病1个月大鼠视网膜内EB浓度是正常组的4.67倍,P<0.01,提示糖尿病大鼠血-视网膜屏障受损。玻璃体腔注射rAAV2-NTF2,视网膜内EB浓度为(8.30±4.16)μg/g视网膜干重,较对侧眼(22.13±8.43)μg/g视网膜干重明显减少,可以部分改善血-视网膜屏障的破坏,降低EB-白蛋白渗漏(P<0.05)。结论:玻璃体腔注射rAAV2-NTF2可减轻糖尿病大鼠视网膜血-视网膜屏障功能的破坏,为糖尿病视网膜病变的基因治疗提供了实验依据。AIM ： To explore the effect of rAAV2 - NTF2 on the blood - retinal barrier damage in early stage diabetic retinopathy. METHODS： Retinal vasculature was observed by Evans blue. NTF2 gene was cloned into adeno - associated virus vector pSNAV. The recombinant pSNAV - NTF2 was transfected into BHK cells. After purification, high - titer rAAV2 - NTF2. rAAV2 - NTF2 at dose of 4μL （titer 1.0× 10^12 ） were injected into left eyes and rAAV2 - EGFP at the same does and titer were injected into the right eyes of 36 rats. After 1 month, diabetes mellitus were induced by STZ. One month after onset of diabetes, EB at a dose of 45 mg/kg was intravenously injected into the rats. Two hours lat- er, both eyes were enucleated immediately after perfusion of 1% PFA -citric acid buffer solution. The retinas were then dissected away and placed in formamide to extract the dye. The concentration of dye was measured by spectrophotometer. RESULTS： Evans blue was contained in normal retinal vessels without leakage, with a very low level of background fluo- rescence. The vessels staining of diabetic rats＇ retina showed increasing fluorescence, indicating the retina - blood barrier damage. Dye concentration, representing the degree of the BRB injury, was higher in retina of 1 month diabetic rats than that in normal rats （4. 67 times, P 〈0. 01 ）, indicating retinablood barrier break -down. Diabetic rats with rAAV2 - NTF2 intravitreal injection showed decreased BRB breakdown （P 〈 0. 05）. CONCLUSION ： High expression of NTF - 2 decreases the BRB dysfunction in DM rats.

关 键 词：糖尿病视网膜病变 核运输因子2 腺相关病毒 血-视网膜屏障 

分 类 号：R363[医药卫生—病理学]