血管内皮生长因子shRNA表达质粒抑制鼠角膜新生血管与血管内皮细胞中血管内皮生长因子的表达

目的 探讨RNA干扰对大鼠碱烧伤后角膜新生血管(CNV)与血管内皮细胞(EC)中血管内皮生长因子(VEGF)表达的影响及作用机制.方法 实验研究.采用DNA重组技术设计合成针对VEGF mRNA的3条shRNA寡核苷酸片段,构建真核表达质粒VEGF shRNA1、2、3,然后将质粒通过脂质体介导分别转染EC,逆转录聚合酶链反应和Western blot免疫印迹法分别检测EC中VEGF mRNA和蛋白的表达及干扰效率,并筛选出干扰效率最高的质粒(VEGF shRNA3质粒)进行体内实验.将20只SD大鼠按完全随机设计方法分为两组,分别为实验组10只和对照组10只,建立大鼠碱烧伤后CNV模型.实验组于结膜下注射VEGF shRNA3质粒后,显微镜下观察CNV生长情况,7 d后利用逆转录聚合酶链反应(RT-PCR)与Western blot法分别检测实验组和对照组角膜组织VEGF mRNA和蛋白的表达.多组间差异显著性检验用单因素方差分析,两组CNV面积比较采用重复测量的方差分析.结果 经酶切及测序证实重组质粒构建成功.VEGF shRNA1、2、3质粒均可下调VEGF mRNA和蛋白的表达,其中VEGF shRNA3质粒的抑制作用较强,VEGF mRNA和蛋白的抑制率分别为68.92%和66.22%.体内实验中碱烧伤后3、5、7 d,实验组CNV长度较对照组明显降低,差异有统计学意义(F=355.744,P=0.000);实验组CNV面积较对照组明显降低,差异有统计学意义(F=402.700,P=0.000);实验组VEGF mRNA和蛋白在角膜组织中的表达较对照组明显降低,抑制率分别为41.84%和41.86%.结论 构建的VEGF shRNA质粒能右效抑制EC中VEGF mRNA和蛋白的表达.结膜下注射VEGF shRNA3质粒能显著抑制碱烧伤后CNV的形成与VEGF的表达.

VEGF short hairpin RNA expression plasmid blocks VEGF expression in rat vascular endothelial cell and inhibits rat corneal neovascularization

Objective To investigate RNA interference in rat corneal neovascularization and vascular endothelial cells of vascular endothelial growth factor expression and its mechanism. Methods In empirical study short hairpin RNA (shRNA) targeting VEGF mRNA was designed and synthesized and VEGF shRNA eukaryotic plasmids were transfected into EC with Lipofectamine 2000 transfection system.Expression of VEGF mRNA and protein in transfected cells was detected by RT-PCR and Western blot.Twenty SD rats were randomized into two groups by randomly Design, experimental group were 10 and 10 in the control group. CNV was induced in vivo by alkaline cauterization of the central cornea, the rats received subeonjunctival injection of VEGF shRNA3 plasmid. Then corneal neovascularization was evaluated with microscope. After seven days, VEGF mRNA and protein was determined by RT-PCR and Western blot respectively. Results VEGF shRNA expression plasmids were successfully constructed and identified by restriction enzyme and sequence analysis. VEGF expression was down regulated by VEGF shRNA1 , 2, 3 plasmids, and VEGF shRNA3 plasmid showed stronger inhibitory effect. The inhibitory effects of VEGF mRNA and protein are 68. 92% and 66.22%. At the 3, 5 and 7 days after alkali cauterization, the CNV length of the experimental group was obviously decreased comparing with the control group, the difference between two groups has statistics meaning( F = 402. 700, P = 0. 000); the CNV area of the experimental group was also obviously decreased comparing with the control group, the difference between two groups has statistics meaning (F =402. 700,P =0. 000); the VEGF mRNA and protein in corneas was also decreased,the inhibitory effects of which are 41.84% and 41.86%. Conclusions RNAi significantly suppresses expression of VEGF in rat EC in vitro. In vivo, subconjunctival delivery of VEGF shRNA3 plasmid suppresses injury-induced VEGF expression and CNV.