结膜下和玻璃体内注射重组人PEDF对氧诱导大鼠RNV的作用

Effects of pigment epithelium-derived factor on oxygen-induced retinal neovascularization in rats with subconjunctival or intravitreal injection

目的:观察结膜下和玻璃体注射重组人色素上皮衍生因子(pigment epithelium-derived factor,PEDF)两种注射方式对氧诱导大鼠视网膜新生血管(retinal neovascularization,RNV)的作用。方法:新生大鼠2只进行左眼结膜下注射PEDF,Westernblot检测视网膜PEDF的表达。氧诱导SD新生大鼠建立类似早产儿视网膜病变动物模型。新生鼠48只随机分为6组(A:空气对照组,B:高氧对照组,C:高氧+玻璃体注射PEDF2μg组,D:高氧+结膜下注射PEDF2μg组,E:高氧+结膜下注射PEDF4μg组,F:高氧+结膜下注射PEDF8μg组)。当新生大鼠脱离氧时,C,D,E和F组大鼠左眼用不同剂量和注药次数进行注射PEDF,ADP酶视网膜血管染色观察视网膜血管形态,石蜡切片计数突破视网膜内界膜的新生血管内皮细胞核数目。结果:结膜下注射重组人PEDF,可检测到视网膜PEDF蛋白表达。视网膜铺片结果显示:A组视网膜血管发育正常;B组视网膜大量的新生血管生成;C组新生血管明显减少;D,E,F组新生血管稍减少。组织病理学检测突破视网膜内界膜的新生血管内皮细胞计数可见:A组视网膜内界膜平滑,偶见突破的视网膜内皮细胞。B组明显高于A组,差异有统计学意义(P<0.05)。C组明显低于B组(P<0.05),D,E,F组与C组差异有统计学意义(P<0.05)。结论:巩膜和脉络膜-色素上皮层对PEDF是有渗透性的,可以跨越结膜下组织到达视网膜,但与玻璃体注射组相比,抑制新生血管作用有显著减弱;玻璃体注射可有效抑制氧诱导大鼠RNV。

AIM：To observe the inhibitive effect of subconjunctival or intravitreal injecting pigment epithelium-derived factor（PEDF） for retinal neovascularization（RNV） in oxygen-induced animal models.METHODS：Mouse models of oxygen-induced retinopathy of prematurity were established.48 newborn rats were randomly diveded into 6 groups（A：Air control group;B：High oxygen control guoup;C：High oxygen ＋intravitreal injection of 2μg PEDF group;D：High oxygen＋subconjunctival injection of 2μg PEDF group;E：High oxygen＋subconjunctival injection of 4μg PEDF group;F：High oxygen＋subconjunctival injection of 8μg PEDF group）.When the rats were taken off the high oxygen,the phosphate buffer solution（PBS） and corresponding dosage of PEDF were intravitreally or subconjunctivally injected in group C,D,E and F respectively.ADP-ase stained retinal flat-mounts was used to evaluate the retinal vascular modality and development,eyeball pathological section was used to count the pre-retinal endothelial nucleus in retinal new vessel.Retinal PEDF expression were detected by Western blot.RESULTS：PEDF were achieved in retina after a periocular injection.Retinal flat-mounts：A group：The retina vessels of the normal had a fine radial branching pattern that extended from the optic nerve to the periphery;B group：There were more RNV compared with A group.The density and shape of RNV were disordered;Compared with B group,RNV decreased obviously in C group while D,E,F group decreased mildly.HE staining：A group：Retinal internal limiting membrane was smooth,and occasionally the endothelial cells broke through the inner retina.B group was significantly more than A group（P0.05）.C group was significantly less than the B group（P0.05）,D,E,F group had significant difference with C group（all P0.05）.CONCLUSION：PEDF are able to penetrate the sclera into retina after a periocular injection,however,compared with intravitreal injection,the effect reduced significantly.How to reach the retina effectively requires further study by subconjunctival injection.