Delta样配体4单克隆抗体对视网膜新生血管形成和血管内皮生长因子表达的影响

The effect of Delta-like ligand 4 monoclonal antibody on retinal neovascularization and vascular endothelialgrowth factor expression

背景 Delta样配体4（Dll4）参与视网膜内细胞的发育和血管的发生过程,并与血管内皮生长因子（VEGF）共同参与诱导和挑选尖端细胞的过程.Dll4在视网膜新生血管形成中的作用及其与VEGF表达的关系值得关注. 目的 研究Dll4单克隆抗体玻璃体腔内注射后对视网膜中Dll4、VEGF及其受体表达和视网膜新生血管形成的影响.方法 将5日龄的新生SPF级SD大鼠与母鼠一起在含体积分数为（80±2）％氧气的密闭玻璃箱内饲养至12日龄,然后返回自然环境下饲养至17日龄以建立大鼠氧诱导视网膜病变（OIR）模型.于模型大鼠12日龄时右眼玻璃体腔内注射Dll4单克隆抗体2.5 μl（0.5 μg）为Dll4单克隆抗体注射组,左眼以同样的方式注射等体积PBS作为PBS对照组.于大鼠17日龄时处死大鼠并分离视网膜.应用逆转录PCR（RT-PCR）法检测两组大鼠视网膜中Dll4、VEGF、VEGF受体-1（VEGFR-1）、VEGFR-2、神经纤维网蛋白-1（neuropilin-1）mRNA的表达情况,采用视网膜铺片ADP酶染色法观察大鼠视网膜新生血管形态,采用视网膜切片苏木精-伊红染色法计数突破内界膜的血管内皮细胞核数目,评估新生血管的严重程度.两组间各检测指标的差异比较采用配对t检验. 结果 Dll4单克隆抗体注射组大鼠视网膜内Dll4 mRNA表达灰度值（Dll4 mRNA/β-actin mRNA）为0.22±0.06,明显低于PBS对照组的0.98±0.13,差异有统计学意义（t=21.839,P=0.000）,而2个组间VEGF mRNA及其受体VEGFR-1 mRNA、VEGFR-2 mRNA表达灰度值的差异均无统计学意义（t=0.463,P=0.649;t=1.687,P=0.109;t=-1.674,P=0.111）；与PBS对照组比较,Dll4单克隆抗体注射组小鼠视网膜中neuropilin-1 mRNA的表达量明显升高,差异有统计学意义（0.73±0.08 vs.0.64±0.07）（t=-2.677,P=0.015）.Dll4单克隆抗体注射组大鼠视网膜新生血管密度明显高于PBS对照组.Dll4单克隆抗体注射组大鼠视网膜突破内界膜的血管内皮细胞数为（63.6±11.6）个/张,PBS对照组为（35.1±5.2）个/张,差异有统计学意义（t=-7.879,P=0.000）. 结论 Dll4在视网膜新生血管形成的过程中发挥重要作用,并可能通过对VEGFR的反馈抑制发挥抑制病理性新生血管过度形成的作用.

Background Studieshowed thaDelta-like ligand 4 （Dll4） participatein the deveopmenof retinal celland angiogenesis.The Dll4-Notch pathway and vasculaendothelial growth facto（VEGF） are thoughto be critical mediatorof neovascularization undehypoxiconditions.The relationship between Dll4 and VEGF inovery cleaand furtheresearch ineeded.Objective Thistudy wato observe the inhibition of Dll4 on experimental retinal neovascularization and VEGF expression.MethodThe retinal neovascularization animal model wainduced by oxygen-induced retinopathy （OIR） in 5-day-old SPF SD ratby rearing the new postnatal ratwith the motherattogethein closed box with oxygen level a（80±2） % till 12-day-old.The ratwere then raised in normal aifo5 days.Aftethat,2.5μl （0.5 μg） of Dll4 monoclonal antibody wainjected into the mid-vitreoucavity in the righeye（Dll4 injected group） and PBwaused in the same way in the fellow eye（PBcontrol group） in the 12-day-old rats.Retinawere isolated in the 17-day-old rats,and retinal vasculamorphology waexamined by adenosine diphosphatease （ADPase） staining of retinal flatmounts,and the endotheliocyte nuclei above the internal limiting membrane were counted in the retinal tissue-slices.Reverse transcription PC（RT-PCR） waused to detecthe mRNexpression level of Dll4,VEGF,VEGF receptor-1 （VEGFR-1）,VEGFR-2 and neuropilin-1 mRNin the retinas.Statistical analysiwaperformed by the paired t-test.The care and use of the animalcomplied with the Guidance Suggestion issued by the Ministry of Science and Technology of Chinin 2006.ResultThe Dll4 mRNexpression in the retin（Dll4 mRNA/β-actin mRNA） wa0.22± 0.06 and 0.98 ± 0.13 in the Dll4 injected group and the PBcontrol group,respectively,with statistically significandifference （=21.839,P =0.000）.No significandifferencewere found in the expression of the VEGF mRNA,VEGFR-1 mRNand VEGFR-2 mRNin the retinabetween the two group（t=0.463,P=0.649;=1.687,P=0.109;=-1.674,P=0.111）.Compared with the PBcontrol group,the expression of neuropilin-1 mRNwasignificantly elevated in the Dll4-injected group （0.73±0.08 vs.0.64±0.07） （t=-2.677,P=0.015）.ADPase staining showed thathere were much more new blood vesselin the Dll4 injected group than those of the PBcontrol group.The numbeof nuclei structurally adjacento the vitreal side of the internal limiting membrane wa（63.6± 11.6）/slide in the Dll4 injected group,which wamore than thaof the PBcontrol group a（35.1±5.2）/slide （=-7.879,P =0.000）.ConclusionDll4 playan essential role in the procesof pathological angiogenesiin the retina.Dll4 ithoughto be feedback regulatoof VEGFR,which participatein the procesof restraining pathological vasculogenesis.