rAAV2-NTF2对糖尿病大鼠血视网膜屏障的保护作用

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4 μL rAAV2-NTF2，左眼为对照眼，玻璃体腔注射rAAV2-增强型绿色荧光蛋白（EGFP）4 μL。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。

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

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 4 μL (titer 1.0×1012) 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 later, 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 fluorescence. 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 retina-blood 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.