肿瘤坏死因子α抑制剂对激光诱导脉络膜新生血管的影响

Effect of anli-TNF-a on laser-induced choroidal neovascularizalion

目的探讨肿瘤坏死因子α（TNF-α）在激光诱导的小鼠脉络膜新生血管（CNV）形成中的作用。方法野生型C57BL／6J小鼠36只（72只眼），每只眼视网膜上做4个光凝斑诱导CNV生成。小鼠分别于光凝前3d或后3d腹腔内植入渗透泵，给予TNF-α抑制剂依那昔普（5μg/h，n=12）或英利西单抗（5μg/h，n=12），持续7d，对照组给予PBS（n=12）。光凝前3d的用药组分别于光凝后1和2周，光凝后3d的用药组于光凝后10d做荧光素眼底血管造影（FFA），之后立刻取左眼行视网膜组织学检查，右眼制作脉络膜平片，免疫组化方法标记内皮细胞，测定荧光素渗漏面积及脉络膜平片CNV相关荧光面积，比较CNV病变的大小及程度。用免疫印迹法分析比较小鼠光凝前后视网膜色素上皮层及脉络膜上TNF-α蛋白的表达。结果光凝后1周，免疫印迹法结果显示小鼠脉络膜及视网膜色素上皮层TNF-α表达增加。光凝前3d用药，光凝后1周，荧光素渗漏面积为（0．74±0．33）×10^4像素，2周时则为（0．63±0．20）×10^4像素，与对照组[（2．61±0．80）×10^4像素]比较均显著减少（t=3．69，3．56；P〈0．05）。光凝后1周对照组、依那昔普组和英利西单抗组CNV面积分别为（3．61±0．93）×10^5、（1．89±0．62）×10^5和（2．14±0．72）×10^5像素，与对照组比较，差异均有统计学意义（t=3．10，2．51；P〈0．05。但光凝后3d用药，则只有依那昔普能显著抑制CNV病变。组织病理学检查进一步证实，治疗组小鼠的CNV病变直径及厚度小于对照组。结论TNF-α参与激光诱导的CNV的形成。TNF-α抑制剂能抑制激光诱导的CNV的形成及渗漏。

Objective To investigate the role of TNF-α in the development of laser-induced choroidal neovascularization （CNV） in the mouse. Methods Laser photocoagulation was used to induce CNV in wild-type C57BL/6J mice by making four separate choroidal burns in each eye. Animals were treated 3 days before or after laser injury with recombinant TNF receptor P75 （etanercept, 5 μg/h, groupl, n = 12）, chimeric monoclonal antibody （ infliximab, 5 μg/h, group2, n = 12） for 7 days by intraperitonealy implanted osmotic pumps. PBS was used as control （ group3, n = 12）. The left eyes were removed for histopatbologic examination and the right eyes were removed for flatmounts immunobistochemistry immediately after fluorescien angiography. In mice treated with medications 3 days before laser injury, left eyes were collected at 1 or 2 weeks after laser injury. In mice treated with medications 3 days after laser injury, left eyes were collected at 10 days after laser injury. CNV responses were compared by flatmount analysis of CNV-related fluorescence area and by determination of fluorescein angiographic leakage. The level of protein expression of TNF-α was semiquantitatively evaluated by Western blot analysis of the choroidal and RPE layer from mice with or without laser treatment. Results Western blotting demonstrated that TNF-α was highly expressed in choroidal and RPE cells of wild type mice 1 week after laser treatment as compared to the control mice without laser treatment. Etanercept and infliximab administrated 3 days before laser-damage significantly reduced CNV size and pathological fluorescein leakage in comparison to the control group one and two weeks after laser injury. Only etanercept administered 3 days after laser injury still significantly reduced the development of CNV lesions. Histopathological examination confirmed that CNV lesions in treated mice had smaller diameter and thinner center as compared to the control animals. Conclusions Anti-TNF-α treatment reduces the size and leakage of laser-induced CNV. These results suggest the involvement of TNF-α in the development of laser-induced CNV and its potential use as a therapeutic agent in the age related maeular degeneration. （Chin J Ophthalmol, 2008,44：200-206）