摘要背景 视网膜新生血管性疾病是一种严重影响视力的眼病.研究表明,cathepsin B参与新生血管的生成,寻找抑制视网膜新生血管形成的药物可为此类疾病的分子机制研究提供依据. 目的 研究cathepsin B-RNAi-lentivirus对小鼠视网膜新生血管的抑制作用. 方法 将7日龄清洁级C57BL/6J小鼠与其母鼠共同置于氧体积分数为(75±2)％的密闭氧箱内5d,制作视网膜新生血管模型,5d后返回正常环境.应用随机数字表法将60只小鼠随机分为模型对照组、阴性对照组和基因治疗组,每组20只.模型对照组小鼠不给予任何药物干预,阴性对照组小鼠玻璃体腔内注射空载体与辅助载体包装成的无目的基因的空病毒颗粒(NC-GFP-LV)1 μl,基因治疗组以同样的方法注射eathepsin B-RNAi-lentivirus 1μl.注射5d后各组分别应用随机数字表法随机处死7只小鼠,获取14只眼的视网膜,采用real-time PCR法检测小鼠视网膜中cathepsin BmRNA的表达量(2△△Ct),另外同时间点同法分别处死8只小鼠,获取16只眼球的视网膜,采用Western blot法检测各组小鼠视网膜中cathepsin B蛋白的相对表达量(cathepsin B/β-actin).注射5d后各组分别取剩余5只小鼠行异硫氰酸葡聚醛荧光素(FITC-dextran)心脏灌注,并处死动物,制备10只眼的视网膜铺片,荧光显微镜下观察和比较各组小鼠视网膜新生血管的形态和数量. 结果 基因治疗组小鼠视网膜中cathepsin B mRNA的表达水平(2-△△Ct)为0.740.12,明显低于模型对照组及阴性对照组的1.66±0.17和1.58±0.29,差异均有统计学意义(q=0.746、1.588,P＜0.01).基因治疗组小鼠视网膜中cathepsin B蛋白的表达水平(cathepsin B/β-actin)为0.64±0.06,明显低于模型对照组及阴性对照组的0.93±0.09和0.96±0.09,差异均有统计学意义(q=0.637、0.894,P＜0.01).小鼠视网膜荧光染色结果显示,模型对照组和阴性对照组小鼠视网膜新生血管分层及分支多,血管走形扭曲,部分血管可见荧光素渗漏；基因治疗组小鼠血管走形较直,血管分支少,新生血管数量少. 结论 Cathepsin B-RNAi-lentivirus转染能有效抑制小鼠视网膜新生血管的形成.

abstractsBackground Retinal neovascularization disease is a group of threatening-vision eye diseases.Researches showed that cathepsin B is involved in angiogenesis.Exploring a drug which inhibit retinal blood vessels will provide the basis for the molecular mechanism of these diseases.Objective This study was to investigate the inhibitory role of cathepsin B-RNAi-lentivirus on retinal angiogenesis.Methods Sixty 7-day-old C57BL/6J mice were raised together with maternal mice in the closed box with the oxygen concentration of (75-2)％ for 5 days to establish the retinal angiogenesis mouse models.The mice were then taken into the normal air environment for continuous raise and were randomized into 3 groups.NC-GFP-LV of 1 μl and the equal volume of cathepsin B-RNAi-lentivirus was intravitreously injected respectively in 40 eyes in the control group and the gene treatment group,and no drug was administered in the 40 eyes of the model group.The mice were sacrificed and retinas were obtained.Expression of cathepsin B protein in the retina was detected by Western blot assay (cathepsin B/β-actin).Real-time PCR was used to detect and compare the expression level of cathepsin B mRNA (2△△Ct).FITC-dextran was used to perform heart infusion for the retinal stretched preparation 5 days after intravitreously injection.Retinal neovascularization was examined by fluorescent angiography.Results The expression level (2-△△Ct) of cathepsin B mRNA was 0.74 ±0.12 in the gene treatment group,showing a significant decline in comparison with 1.66±0.17 and 1.58±0.29 in the model group and control group (q--0.746,1.588,P＜ 0.01).The expression level of cathepsin B protein (cathepsin B/β-actin) in the retina was 0.64±0.06,0.93±0.09 and 0.96±0.09 respectively in the gene treatment group,model group and control group,indicating a significant reduce in the gene treatment group (q =0.637,0.894,P＜0.01).Distorted vessels were seen in the mice retinas of the model group with more branches and vascular anastomosis,and fluorescine leakage was exhibited under the fluorescence microscope.However,the vessels were regular with less branches and angiogenesis.Conclusions Cathepsin B-RNAi-lentivirus can effectively inhibit oxygen-induced retinal angiogenesis in mouse.