雷珠单抗对角膜新生血管的抑制作用以及对血管生成相关miRNAs表达的影响

Effects of ranibizumab on corneal neovascularization and angiogenesis-related miRNAs expression in rats

目的探讨雷珠单抗对角膜新生血管(corneal neovascularization,CNV)的抑制作用以及对血管生成相关miRNAs表达的影响。方法取24只SD大鼠随机分为3组,左眼角膜缝线法制备CNV模型后分为模型组和治疗组(结膜下注射10 g·L^-1雷珠单抗注射液),每组各8只,剩余8只作为空白组。术后第8天评估大鼠CNV长度及面积,行组织病理学检查及CD31免疫荧光染色。检索基因芯片与生物信息学数据库,选择血管生成相关的miRNAs,实时荧光定量PCR验证各组VEGF-A及miRNAs的表达。采用生物信息学方法分析miRNA靶基因富集通路。结果治疗组大鼠CNV长度、面积均小于模型组,差异均有统计学意义(均为P<0.01)。HE染色显示,治疗组大鼠角膜仅有少量新生血管及炎症细胞,模型组大鼠角膜中出现大量新生血管且各层均可见炎症细胞。CD31免疫荧光染色显示,模型组及治疗组中角膜均可见DAPI阳性染色,CD31主要在基质层表达,形成环形管腔结构。模型组CD31染色阳性的每个视野微血管数为(9.83±1.85)个(400倍),治疗组中每个视野微血管数为(4.58±1.38)个,3组角膜微血管数差异有统计学意义(F=163.65,P<0.01)。治疗组VEGF-A mRNA表达水平低于模型组,miR-15b、miR-16、miR-29c表达水平均高于模型组,差异均有统计学意义(均为P<0.01)。差异miRNAs的靶基因主要富集于血管生成、蛋白结合等通路。结论结膜下注射雷珠单抗能抑制大鼠CNV,降低VEGF-A表达并改变相关miRNAs表达水平。

Objective To investigate the effects of ranibizumab on corneal neovascularization(CNV)and angiogenesis-related miRNAs expression in rats.Methods A total of 24 SD rats were randomly divided into three groups,and corneal suture was conducted on the left eyes of 16 rats in order to induce the development of corneal neovascularization and later these rats were divided into model group and treatment group(subconjunctival injection of 10 g·L^-1 ranibizumab),and the remaining 8 rats were classified as the blank group.The length and area of CNV of rats were calculated on the 8th day after surgery.Then the morphological structure of corneal was observed by HE staining and the expression levels of CD31 were detected by using immunofluorescence staining.Expression levels of VEGF-A and miRNAs were detected by RT-PCR.The target genes of miRNA were analyzed by bioinformatics methods.Results On the 8th day after surgery,the length and area of CNV in the treatment group were significantly smaller than those in the model group(both P<0.01).Histopathological examination showed the presence of a large number of inflammatory cells and neovascularization in the corneal tissue of the model group whereas only a small number could be detected in the treatment group.CD31 immunofluorescence staining showed that DAPI-positive staining was seen in the cornea in the model group and the treatment group,and CD31 was mainly expressed in the stroma,forming a circular lumen structure.Immunofluorescence showed that the number of CD31-positive microvessels in the treatment group(4.58±1.38)was lower than that in the model group(9.83±1.85)(F=163.65,P<0.01).The expression of VEGF-A mRNA in the treatment group was down-regulated compared with the model group,while the expression levels of miR-15b,miR-16,and miR-29c were up-regulated(all P<0.01).Target genes of differently expressed miRNAs were mainly enriched in angiogenesis,protein binding items.Conclusion Subconjunctival injection of ranibizumab can reduce the formation of CNV,decrease the level of VEGF-A and change the level of related miRNAs.