ADP-核糖基化因子抑制剂在实验性碱烧伤诱导角膜新生血管中的作用及其机制

背景角膜新生血管（CNV）是导致角膜盲的主要原因之一，研究表明，ADP一核糖基化因子（ARF）对肿瘤细胞的增生有调节作用，抑制ARF能抑制血管的形成，但ARF抑制剂对CNV是否发挥作用尚不清楚。目的探讨ARF抑制剂在碱烧伤诱导的CNV形成过程中的作用及其机制。方法7～8周龄清洁级雄性BABL／c小鼠60只按照随机数字表法分为PBS对照组和ARF抑制剂干预组，每组各30只。所有小鼠采用角膜碱烧伤诱导法构建CNV模型，ARF抑制剂干预组小鼠于造模后1周腹腔内注射0．5g／LARF抑制剂溶液0．5ml，每周3次，共1周，PBS对照组小鼠以同样方式注射0．5mlPBS。各组分别取24只小鼠，于造模后0、4、7、14d采用实时定量PCR（real—timePCR）和Westernblot法检测小鼠角膜组织中ARFmRNA和蛋白的动态表达，并于造模后14d，采用Westernblot法检测各组小鼠角膜组织中血管内皮生长因子（VEGF）的表达。各组另取6只小鼠，于造模后2、4、7、14d裂隙灯显微镜下观察CNV形成情况，并于造模后14d，采用全视网膜铺片免疫荧光组织化学法检测角膜组织中CD31在CNV中的表达。体外实验中应用ARF抑制剂干预人视网膜血管内皮细胞（RECs），CCK8法和细胞划痕法检测ARF对人RECs增生和迁移的影响。实验动物的使用和喂养遵循视觉及眼科学研究协会有关规定及苏州大学《实验动物管理及使用指南》。结果造模后PBS对照组和ARF抑制剂干预组小鼠角膜组织中ARFmRNA在各个时间点均有表达，在造模后14d达高峰，各组小鼠角膜中ARFmRNA的表达随着时间的延长而增加，差异有统计学意义（F时间=65．17，P=0．00），不同时间点的组间比较差异无统计学意义（F＊＊=1．98，P=0．18）；造模后14d，ARF抑制剂干预组实验后不同时间点ARF蛋白水平相对表达值比较差异有统计学意义（F=10．77，P=0．00）。裂隙灯显微镜下检查发现，ARF抑制剂干预组CNV相对面积为0．45±0．05，PBS对照组为0．72±0．11，2个组间差异有统计学意义（t=-3．87，P〈0．05）。全角膜铺片免疫荧光组织化学法检测表明，ARF抑制剂干预组小鼠角膜组织CD31表达面积明显小于PBS对照组。造模后14d，Westernblot法检测显示，ARF抑制剂干预组VEGF蛋白表达水平为1．20＋0．21，明显低于PBS对照组的2．47±0．33，差异有统计学意义（t=-5．62，P〈0．05）。CCK8法检测结果显示，随着ARF抑制剂质量浓度的增加，ARF抑制剂对人RECs的抑制率增加，差异有统计学意义（F=8．47，P=0．02）。细胞划痕实验后24h，100μg／L和1000μg／LARF抑制剂干预组人RECs迁移距离分别为（5．46±1．32）μm和（5．04±1．68）μm，与PBS对照组的（8．49±1．18）μm相比明显减小，差异均有统计学意义（t=-2．94、-2．91，P〈0．05）。结论ARF抑制剂能抑制碱烧伤诱导的CNV发生，可能与其下调VEGF的表达以及抑制血管内皮细胞的增生和迁移有关。

The effect of ADP-ribosylation factor antagonist on alkali-burn induced corneal neovascularization

Background Corneal neovascularization （CNV） is one of the causes of corneal blindness. Studies showed that ADP-ribosylation factor （ARF） can regulate the growth of tumor cells,and inhibiting ARF will decrease angiogenesis. However,whether ARF antagonist plays an action on CNV is unclear. Objective The aim of this study was to explore the effect and mechanism of ARF inhibitor on alkali-burn induced CNV. Methods Sixty clean male BABL/c mice aged 7-8 weeks were divided into PBS group and ARF antagonist group according to randomized number table. CNV models were induced by NaOH burn method in all the mice. ARF at the concentration of 0.5 g/L（0.5 ml） was intraperitoneally injected 3 times per week for 1 week followed the induction of CNV in the ARF antagonist group, and 0.5 ml PBS was used in the PBS group. CNV was examined 2,4,7,14 days after injection by the slit lamp microscope and the CNV related area in the cornea was calculated. Before modeling（0 day） and 4,7, 14 days after modeling, real-time PCR and Western blot were used to analyze the expressions of ARF mRNA and protein in the corneas. Forteen days after modeling, the expression of the CD31 in the CNV was detected using immnofluorescence of corneal whole mount;the expression of vascular endothelial growth factor（VEGF） in the cornea was assayed by Western blot. Cellular wound scratch test was employed to evaluate the effects of ARF antagonist on proliferation and migration of human retinal vascular endothelial cells （ RECs）. All animal experiments were done in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and Guideline for the Care and Use of Laboratory Animals on the the Soochow University Animal Care Committee. Results ARF mRNA and protein were expressed in the mice corneas in both the PBS group and the ARF antagonist group at various time points. The expression of ARF mRNA in mice corneas was enhanced with the lapse of the time （ Ftime = 65. 17, P = 0.00） , but no significant difference was found among the groups （Froup = 1.98 ,P = 0. 18 ）. There was also significant difference in the expression of ARF protein in mice corneas at different time points in the ARF antagonist group （ F = 10. 77 ,P=0.00）. The related CNV area was 0.45±0. 05 in the ARF antagonist group,and that in the PBS group was 0.72±0. 11 ,with significant difference between them （t =-3.87,P〈0.05 ）. The green fluorescence area of CD31 expression in the cornea was smaller in the ARF antagonist group than that of the PBS group. Expression level of VEGF in the ARF antagonist group was 1.20±0. 21 ,and that in the PBS group was 2.47±0.33 ,showing a significant difference （ t = - 5.62, P 〈 0.05 ）. As the increase of ARF antagonist concentration, the inhibiting rate of cell proliferation was reinforced among 10,100 and 1 000 t~g/L ARF antagonist groups （ F = 8.47, P = 0.02 ）. Twenty-four hours after scratch test,the migrating distance of human RECs was （5.46±1.32）μm and （5.04±1.68） μm in the 100μg/L and 1 000 μg/L ARF antagonist groups, respectively,which were shorter than （8.49± 1. 18 ） μm of the PBS group （ t = -2.94,-2.91 ,both at P〈0. 05 ）. Conclusions ARF inhibitor can reduce CNV by down-regulating the expression of VEGF in alkaline burn cornea and inhibiting the proliferation and migration of vascular endothelial ceils.