辐射及环孢素对高危角膜移植术后角膜血管内皮生长因子表达的抑制作用

目的探讨电离辐射对高危角膜移植术后血管内皮生长因子（VEGF）表达及角膜新生血管（CNV）形成的抑制作用。方法用角膜缘缝线法刺激CNV,将15只SD大鼠的角膜作为供体、30只Wistar大鼠的右眼作为受体行穿透角膜移植术。将受体大鼠随机分为3组,每组10只眼。A组为辐射组,术后第1天β射线7Gy照射右眼,每日1次,共7次;B组为质量分数1%环孢素A（CsA）组,术后第1天起点用1%CsA滴眼液,每日3次,共7d;C组为单纯移植组。每日裂隙灯下观察角膜植片的临床反应。分别于干预后7d、14d摘除鼠眼获得角膜标本,采用免疫组织化学法检测VEGF蛋白在角膜中的阳性表达情况,Westernblot方法半定量观察VEGF蛋白在角膜中的表达,逆转录聚合酶链反应（RT-PCR）法检测VEGFmRNA在各组角膜组织中的表达。结果裂隙灯下可见角膜移植后14d,与C组比较,A组和B组角膜植片水肿较轻,CNV较少。免疫组织化学检测表明A组和B组VEGF在角膜组织中的表达呈淡黄色,C组呈棕色。RT-PCR检测显示,VEGFmRNA的反应条带在C组最强、A组最弱,Westernblot检测的结果与RT-PCR相同。结论 90Sr-90Y眼科敷贴器小剂量分次放射治疗及1%CsA滴眼液局部应用可明显抑制高危角膜移植术后新生血管的生长,90Sr-90Y眼科敷贴器辐射作用强于1%CsA滴眼液。

Inhibitory effect of iridition and cyclosporin A on vascular endothelial growth factor expression induced by high risk keraplasty

Background Although corneal tissue is a immunologically privileged site,but the immunity rejection is frequently occurred after corneal transplantation,especially the cornea with high risk factor.Objective The present project was to approach the inhibiting effect of radiation on the expression of vascular endothelial growth factor （VEGF） and corneal new vessels after high risk keratoplasty.Methods High risk keratoplasty models were established by suturing in corneal limbus to stimulate the new vessels of cornea in the right eyes of 30 receipts of clean Wistar rats prior to the penetrating corneal transplantation,and then the grafts from donors of 15 clean SD rats was implanted into the receipt eyes.90Sr-90Y pather with the dose of 7 Gy was used to irradiate the 10 model eyes once per day for 7 days in irradiation group,and 1% cyclosporin A （CsA） eye drop was topically administered in other 10 model eyes three times per day for 7 days in 1%CsA group from the first day following the keratoplasty.The 10 model eyes only receiving keratoplasty worked as the positive control group.Status of grafts was clinically evaluated under the slim lamp.The animals were sacrificed and corneas were obtained from the models at 7 and 14 days after intervened.The expression of vascular endothelial growth factor （VEGF） in cornea after irradiation of 90Sr-90Y and administration of 1%CsA eye drop was detected by immunohistochemistry,and the expressions of VEGF and VEGF mRNA were semiquantitatively detected by RT-PCR and Western-blot at the same time points.The use of animals followed the Statement of ARVO.Results The mild edema and opacification of cornea were seen on 14 days in both radiation and 1%CsA group,and CNV was less in comparison with positive control group under the slim lamp.The expression of VEGF protein was mainly in vascular endothelial cells,corneal epithelial layer and stroma layer,showing the slight yellow granules,yellow granules and brown granules on 14 days in irradiation group,1%CsA group and positive control group.The infiltration of a lots of inflammatory cells was seen in positive control group.RT-PCR result showed that the expression bands of VEGF mRNA was strongest in positive control group and weakest in irradiation group,and Western blot result revealed the same pattern among the three groups.Conclusion Low dose radiation of 90Sr-90Y ophthalmic applicator and 1%CsA can inhibit corneal neovascularization after high risk keratoplasty.90Sr-90Y ophthalmic applicator has the stronger effect than 1%CsA eye drop.