组织蛋白酶B和血管内皮生长因子在高氧诱导的视网膜新生血管中的作用

背景 血管内皮生长因子(VEGF)在血管发育和新生血管形成中起着关键作用.研究表明,组织蛋白酶B(Cathepsin B)参与新生血管的生成,但其具体机制尚不明确. 目的 探讨Cathepsin B和VEGF在高氧诱导小鼠视网膜新生血管中的表达及二者之间的关系. 方法 应用随机数字表法将44只7日龄清洁级C57BL/6J小鼠随机分为正常对照组、高氧诱导组、空白对照(NC)-绿色荧光蛋白(GFP)-慢病毒(Lv)组和Cathepsin B-RNA干扰(RNAi)-Lv组,每组11只小鼠22只眼.正常对照组小鼠在自然环境中生长;其余3个组7日龄小鼠置于氧体积分数为(75±2)％的密闭氧箱内饲养5d,之后返回到正常环境中.高氧诱导组的12日龄小鼠不给予任何药物干预;NC-GFP-Lv组和Cathepsin B-RNAi-Lv组的12日龄小鼠分别给予玻璃体腔注射NC-GFP-Lv和Cathepsin B-RNAi-Lv各1μl.取各组17日龄小鼠,颈椎脱臼法处死后剥取视网膜,分别采用real-time PCR和Western blot法检测小鼠视网膜中Cathepsin B和VEGF mRNA及蛋白的相对表达量.结果 荧光显微镜下Cathepsin B-RNAi-Lv组视网膜新生血管分层和分支均较高氧诱导组和NC-GFP-Lv组少.各组Cathepsin B和VEGF mRNA总体比较,差异均有统计学意义(F=444.89,P=0.00;F=519.78,P=0.00),其中高氧诱导组、NC-GFP-Lv组和Cathepsin B-RNAi-Lv组小鼠视网膜中Cathepsin B和VEGFmRNA的相对表达量均明显高于正常对照组,Cathepsin B-RNAi-Lv组小鼠视网膜中Cathepsin B和VEGF mRNA的相对表达量均明显低于高氧诱导组和NC-GFP-Lv组,差异均有统计学意义(均P＜0.05).各组Cathepsin B和VEGF蛋白总体比较,差异均有统计学意义(F=54.37,P=0.00;F=79.65,P=0.00),其中高氧诱导组、NC-GFP-Lv组和Cathepsin B-RNAi-Lv组小鼠视网膜中Cathepsin B和VEGF蛋白的相对表达水平均明显高于正常对照组,Cathepsin B-RNAi-Lv组小鼠视网膜中Cathepsin B和VEGF蛋白的相对表达水平均明显低于高氧诱导组和NC-GFP-Lv组,差异均有统计学意义(均P＜0.05). 结论 高氧诱导的视网膜新生血管中Cathepsin B和VEGF高表达,Cathepsin B-RNAi-Lv可以抑制Cathepsin B和VEGF mRNA和蛋白的表达水平,VEGF表达的改变可能是受Cathepsin B表达的影响.

Effect of Cathepsin B and vascular endothelial growth factor on the retinal neovascularization induced by hyperoxia

Background Vascular endothelial growth factor (VEGF) plays a key role in the vascular development and neovascularization.Studies have proved that Cathepsin B is related to the formation of neovascularization,but its mechanism is unclear.Objective This study was to investigate the expression of Cathepsin B and VEGF in the retinal neovascularization induced by hyperoxia and the relationship between them.Methods Forty-four 7-day-old C57BL/6J mice were randomly assigned into 4 groups:normal control group,hyperoxia-induced group,normal control (NC)-green fluorescent protein (GFP)-lentivirus (Lv) group and Cathepsin B-RNA interference (RNAi)-Lv group,with 11 mice 22 eyes for each group.The mice in normal control group were survival in natural environment.The other three groups of 7-day-old mice were put in a sealed box of oxygen volume fraction (75±2)％ for 5 days and then sent back to normal environment.The mice of hyperoxia-induced group did not have any drug intervention,while the 12-day-old mice of NC-GFP-Lv group and Cathepsin B-RNAi-Lv group received intravitreal injection of NC-GFP-Lv 1 μl or Cathepsin B-RNAi-Lv 1 μl.All 17-day-old mice were sacrificed and the retinas were collected.The mRNA expression levels of Cathepsin B and VEGF were performed by real-time PCR;the protein expression levels of Cathepsin B and VEGF were detected by Western blot.Results Fluorescence microscope results showed that the layer and branch of retinal neovascularization were less in the Cathepsin B-RNAi-Lv group than those in the NC-GFP-Lv group and hyperoxia-induced group.The relative expression levels of Cathepsin B and VEGF mRNA in each group were significantly different (F =444.89,P =0.00;F =519.78,P =0.00).The relative expression levels of Cathepsin B and VEGF mRNA in hyperoxia-induced group,NC-GFP-Lv group and Cathepsin B-RNAi-Lv group were higher than those in normal control group,and the relative expression levels of Cathepsin B and VEGF mRNA in Cathepsin B-RNAi-Lv group were lower than those in hyperoxia-induced group and NC-GFP-Lv group,with significant differences between them (all at P ＜ 0.05).The relative expression levels of Cathepsin B and VEGF protein in each group were significantly different (F =54.37,P =0.00;F =79.65,P =0.00).The relative expression levels of Cathepsin B and VEGF protein in hyperoxia-induced group,NC-GFP-Lv group and Cathepsin B-RNAi-Lv group were higher than those in normal control group,and the relative expression levels of Cathepsin B and VEGF protein in Cathepsin B-RNAi-Lv group were lower than those in hyperoxia-induced group and NC-GFP-Lv group,with significant differences between them (all at P＜0.05).Conclusions The expression levels of Cathepsin B and VEGF in retinal neovascularization of hyperoxia-induced group were significantly higher than those in normal control group,Cathepsin B-RNAi-Lv can inhibit the mRNA and protein expression of Cathepsin B and VEGF,the expression of VEGF may be influenced by the expression of Cathepsin B.