目的:探讨以重组腺相关病毒(rAAV)介导内皮抑素(ES)基因转移对大鼠角膜新生血管(corneal neovascularization,CNV)的抑制作用。方法:选取SPF级SD大鼠60只,角膜缝线法制作CNV模型,随机分为A,B,C和D共4组。A组:缝线后立即于上方近角膜缘处结膜下注射50μL rAAV-ES转染液;B组:缝线后刮除角膜上皮,用rAAV-ES转染液浸泡角膜10min;C组:缝线后用rAAV-ES转染液浸泡去上皮角膜10min后,同样部位结膜下注射50μL rAAV-ES转染液;D组:对照组,同样部位结膜下注射50μL生理盐水。在裂隙灯显微镜下观察各组CNV的生长情况,并计算其面积;免疫组织化学法观察ES基因表达情况;病理切片检测CNV密度;观察ES基因转移对CNV的抑制作用。结果:裂隙灯显微镜下观察各组大鼠缝线后CNV逐渐生长,14d达高峰,其后逐渐减少;21d后变性。定量数据重复测量的方差分析显示:时间因素与治疗因素之间存在交互效应(F=175.810,P<0.01)。缝线诱导后不同转染组间CNV面积亦有显著性差异(F=2243.816,P<0.01);其中以D组CNV面积最大;C组CNV面积最小。不同时间段CNV生长面积之间差异显著(F=1060.854,P<0.01);对CNV增生的抑制率随着转染时间的延长而增加,C组在缝线后28d,对CNV增殖的抑制率高达到58.25%。免疫组织化学检测显示:A组于上方角膜缘结膜和角膜上皮中可见ES染色阳性;B组在浅基质CNV的内皮细胞中发现少量ES染色阳性;C组角膜上皮及浅基质CNV的内皮细胞中可见较为明显的ES染色阳性;D组角膜细胞均为ES染色阴性。角膜切片结果显示:CNV密度在各转染组均比对照组稀疏。结论:rAAV-ES转基因可明显抑制缝线诱导的大鼠CNV的增生。采用单独结膜下注射rAAV-ES转染液或转染液局部浸泡去上皮角膜的转染途径均能抑制缝线诱导的CNV增生,联合转染途径对缝线诱导CNV增生的抑制效果更好。

AIM:To evaluate the effect of adeno-associated virus (rAAV)mediated encoding endostatin(ES) gene transfection for inhibiting experimental corneal neovascularization (CNV).METHODS:Sixty healthy SD rats were sutured on the superior cornea.Animals were randomly divided into 4 groups(A,B,C,D).50μL of rAAV-ES solution was injected into subconjunctiva immediately after sutured in the group A.After scraping off epithelium,the cornea was soaked in the rAAV-ES solution for 10minutes in the group B.After scraping off epithelium,the cornea was soaked in the rAAV-ES solution for 10minutes and 50μL of rAAV-ES solution was injected into subconjunctiva in the group C.50μL of saline was injected into subconjunctiva in the group D.Eyes were examined by a slit-lamp biomicroscope and a surgical microscope was used to monitor angiogenesis in response to rAAV-ES transduction.The size of CNV area was measured and calculated.The ES gene expression in inflammatory cornea of rats was detected by immunohistochemistry after transduction with rAAV-ES.The sections were stained with hematoxylin and eosin to measure the CNV density.RESULTS:The occurrence and development of CNV were observed by slit-lamp microscope after transduction with rAAV-ES.After suturing,CNV increased gradually,peaked on day 14,and decreased gradually,degenerated on day 21 after suture induction.Analysis of variance of repeated data showed that there was crossover effect of time and treatment factors(F=175.810,P<0.01).At each transducted group,there was a significant difference between any two groups(F=2243.816,P<0.01).The size of CNV areas of group C was significantly smaller than that of the group A,B and D at each time point.Accordingly,the size of CNV areas of group D was the largest,but the size of CNV areas of group C was the smallest after transduction with rAAV-ES.At each time point,there was a significant difference between any two groups(F=1060.854,P<0.01).Inhibition ratio of CNV was increased gradually after transduction,and the highest inhibition ratio of CNV was detected in group C on day 28 after suture induction.CNV density was lower in transducted group than in control group.The ES gene expression in inflammatory cornea of rats was detected by immunohistochemistry after transduction with rAAV-ES.Only corneal epithelium was stained brown in group A,some of corneal epithelium and NV endothelial cells in superficial stroma of cornea were stained brown in group B,and most of corneal epithelium and NV endothelial cells in superficial stroma of cornea were stained brown in group C,but corneal cells were not stained at all in group D.The sections were stained with hematoxylin and eosin to measure the CNV density.CNV density was lower in transducted group than in control group.CONCLUSION:There was a significant inhibitory effect on neovascularization in rat inflammatory CNV model after rAAV-ES transduction.The inhibitory effect on CNV was higher by use of combined delivery approaches than by use of either one,i.e.the cornea was soaked in the rAAV-ES solution after scraping off epithelia or the rAAV-ES viral particles were injected into subconjunctiva.

R772.2

国家自然基金青年资助项目(No.30901650);中国广东省自然基金资助项目(No.10451051501005773)~~