Background It has been proved that as an important adhesion protein of extracellular matrix,osteopontion (OPN) can affect tumor neovascularization.Some new researches showed that anti-OPN antibody plays a role in regulating the neovascular vessel formation.Choroidal neovascularization (CNV) has the same structure with tumor neovascularization,but whether anti-OPN antibody restricts new vessel formation is unclear.Objective This study was to investigate the inhibitory effect of anti-OPN antibody on CNV.Methods Laser-induced CNV models were created in 40 eyes of 40 male SPF C57BL/6J mice by Argon laser photocoagulation of retinas,with the wavelength 514 nm.Thirty-six successful models were randomly divided into anti-OPN antibody group,mouse-IgG group and PBS group by the randomized number table.On the second day after photocoagulation,anti-OPN antibody of 400 μg was intraperitoneally injected in the anti-OPN antibody group,and the equivalent amount of mouse IgG and PBS were used in the same way in the mouse IgG group and PBS group.The CNV was evaluated by fundus fluorescein angiography (FFA) on the seventh days after photocoagulation.The mice were immediately sacrificed and the eyeballs were enucleated on the fourteenth day after photocoagulation,and 4 eyeballs in each group were used to observe the areas of CNV on the retinal pigmental epithelium-choroid-sclera fiat mounts,and the other 8 eyeballs of each groups were used to analyze the expression levels of OPN mRNA and vascular endothelial growth factor(VEGF) mRNA using quantitative fluorescence-PCR (QF-PCR).Results FFA showed fluorescein leakage areas around laser spots 7 days after photocoagulation,indicating that CNV appeared.The CNV areas were ([16.98±0.70] × 103) μm2,([27.13 ± 0.81] × 103) μm2 and ([35.39±2.14] ×103) μm2 respectively in the anti-OPN antibody group,mouse IgG group and PBS group,with a significant difference among the 3 groups (F =533.76,P =0.00),and the CNV area was significantly smaller in the anti-OPN antibody group compared with those of the mouse IgG group and PBS group (q =-3.95,-4.40,both at P＜0.05).No significant difference was found in the OPN mRNA expression between the antiOPN antibody group and mouse IgG group (t =-5.26,P =0.66).However,the expression of VEGF mRNA in choroidal tissue was significantly declined in the anti-OPN antibody group than that in the mouse IgG group (t =-6.74,P＜0.01).Conclusions Anti-OPN antibody suppresses the formation of CNV in laser-induced mouse model by down-regulating VEGF.%背景 骨桥蛋白(OPN)是细胞外基质中的一种黏附蛋白,可促进肿瘤新生血管生成,且新近研究发现抗OPN抗体可抑制肿瘤新生血管的生成.脉络膜新生血管(CNV)与肿瘤新生血管类似,但抗OPN抗体对CNV形成的作用及机制尚不清楚. 目的 探讨抗OPN抗体对激光诱导的小鼠CNV的干预作用及机制.方法 选取SPF级C57BL/6J雄性小鼠40只,用氩激光(514 nm)视网膜光凝法建立小鼠CNV模型.采用随机数字表法将造模成功的36只模型鼠随机分成3个组,分别于光凝后第2天腹腔内注射兔抗小鼠OPN抗体、抗小鼠IgG和PBS各400 μg.模型鼠于光凝后第7天行荧光素眼底血管造影(FFA)检查;光凝后第14天摘除小鼠眼球,每组中任意取4只眼行视网膜色素上皮-脉络膜-巩膜铺片,行荧光抗体染色观察和CNV定量分析;其他8只眼分离脉络膜后用荧光定量PCR法检测各组小鼠脉络膜中OPN mRNA及血管内皮生长因子(VEGF) mRNA的表达,以吸光度(A)表示.结果 FFA检查显示,光凝后第7天可见光斑周围荧光素渗漏,证实CNV已形成.脉络膜铺片荧光显微镜观察结果显示,在激光光斑处可见绿色团状强荧光影,形态如烟花爆破样,证实光凝处CNV形成.根据脉络膜铺片测得抗OPN抗体组、小鼠IgG组和PBS组小鼠CNV面积分别为[(16.98±0.70) ×103] μm2、[(27.13±0.81) ×103] μm2和[(35.39±2.14) ×103] μm2,3个组间差异有统计学意义(F=533.76,P=0.00),其中抗OPN抗体组CNV的面积明显小于小鼠IgG组和PBS组,差异均有统计学意义(q=-3.95、-4.40,P＜0.05).荧光定量PCR检测显示,抗OPN抗体组与小鼠IgG组间小鼠脉络膜中OPN mRNA表达量的差异无统计学意义(t=-5.26,P=0.66),而抗OPN抗体组小鼠脉络膜中VEGFmRNA的表达量明显低于小鼠IgG组,差异有统计学意义(t=-6.74,P＜0.01). 结论 腹腔内注射抗OPN抗体可下调激光诱导的CNV小鼠模型脉络膜中VEGF的表达,抑制CNV的形成.
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