贝伐单抗对小鼠单纯疱疹病毒性角膜炎角膜新生血管和瘢痕形成的抑制作用

Inhibitory effects of bevacizumab on corneal neovascularization and scarring in mice with herpes simplex keratitis

背景单纯疱疹病毒性角膜炎（HSK）可诱导发生角膜新生血管和炎症反应，传统的治疗药物为阿昔洛韦（ACV），研究已证实贝伐单抗具有抑制新生血管的作用，但其是否对HSK发挥治疗作用值得研究。目的研究贝伐单抗对小鼠HSK角膜瘢痕和新生血管的抑制作用。方法利用体外培养并感染的Vero细胞生产单纯疱疹病毒I型（HSV-1），以无血清DMEM培养基于冰上对HSV．1进行10倍梯度稀释后制备成HSV-1液。选用SPF级雄性6～8周龄C57BL／6小鼠200只，用0．6μl滴度为1×10空斑形成单位（PUF）／ml的HSV-1行小鼠角膜基质注射以制备HSK模型，将模型眼分为单纯ACV注射组、ACV＋贝伐单抗注射组和生理盐水注射组，按照分组分别于感染后5、8、11和14d选择角膜混浊评分为1分的模型眼结膜下注射50μgACV、50IxgACV＋5／xl贝伐单抗和5斗l生理盐水。此外采用紫外线照射均有轻度新生血管和瘢痕的6只模型小鼠双眼诱导HSK复发，于复发后0、2、4和6d行5μl贝伐单抗（25mg／m1）右眼结膜下注射，左眼结膜下注射5μl生理盐水。于造模后5、7、11、14和17d以及复发的O、2、4和6d行小鼠角膜裂隙灯显微镜检查并用角膜知觉测量仪行中央角膜敏感度检测；制备角膜铺片，采用免疫荧光检测法检测角膜中CD31和βⅢTubulin荧光表达以评估角膜新生血管和角膜神经纤维分布；采用ImageJ软件测定角膜新生血管面积和瘢痕面积。结果HSK成模率达80％以上，造模后7d和复发后2d角膜混浊最重，造模后15d和复发后2d角膜新生血管面积最大。造模后模型眼中央角膜敏感度逐渐降低，于造模后9d下降到最低。ACV＋贝伐单抗注射组角膜病变面积小于单纯ACV注射组，ACV＋贝伐单抗注射组小鼠中央角膜敏感度为5．50±O．71，明显高于生理盐水注射组的0．50±1．41，差异有统计学意义（Z=-2．397，P=0．029）。ACV＋贝伐单抗注射组小鼠角膜病变面积增长率为（167．10±52．53）％，低于生理盐水注射组的（312．30±74．18）％，差异有统计学意义（Z=-1．992，P=0．046）。实时荧光定量PCR显示造模后7d，模型眼角膜及同侧三叉神经节（TG）中胸苷激酶（TK）和感染细胞蛋白一27（ICP一27）mRNA相对表达量均较高，造模后45d明显下降，诱导复发后2dTKmRNA和ICP-27mRNA均再次升高，复发后7d表达量降至最低。造模后45d同侧TG中均可见LATmRNA表达量达峰值，诱导复发后2d相对表达量下降，复发后7d相对表达量再次升高，差异均有统计学意义（均P〈O．01）。角膜铺片结果显示，造模后生理盐水注射组小鼠较正常对照小鼠角膜新生血管明显增加，角膜神经纤维明显减少，单纯ACV注射组和ACV＋贝伐单抗注射组小鼠角膜新生血管少于生理盐水注射组，ACV＋贝伐单抗注射组小鼠角膜神经纤维较生理盐水组注射组和单纯ACV注射组均增加。结论贝伐单抗结膜下注射可抑制HSK模型小鼠角膜新生血管生成和瘢痕形成，与ACV联合应用时二者有协同作用。

Background Corneal neovascularization and inflammation occur in herpes simplex keratitis （HSK）. Acielovir （ACV） is an antiviral medication which is primarily used for the treatment of HSV infection. Bevacizumab is an angiogenesis inhibitor which has the ability to slow the growth of corneal neovascularization. However,whether bevacizumab play treating effects on HSK is worth studing. Objective This study attempted to study the effects of bevacizumab on cornea lesion in mouse models of HSK. Methods The solution containing herpes simplex virus type-1 （HSV-1） of Mckrae strain was induced by cultured and infectious ero cells and prepared by ten-times step dilution with free-serum DMEM,and plaque assay was used to detect the viral titers. HSV-1 of 1×107 plaque-forming unit （PFU） in 0. 6 μl was injected into the corneal stroma of 6 to 8- week-old SPF male C57BL/6 mice using a microliter syringe to establish latent HSK mouse models. The models were examined under the slit lamp microscope at day 5,7,11,14 and 17 after modeling as well as day 0,2,4 and 6 after recurrence, and the central cornea touch sensitivity was recorded. The models were divided into ACV-injected group, ACV＋bevacizumab- injected group and normal saline-injected group, and 5 ~1 normal saline with 50 μg ACV, 50 Ixg ACV ＋ 5 μl bevacizumab or 10μ1 normal saline was subconjunctivally injected according to grouping in 4 eyes of each group, respectively. Twelve model eyes were exposed to ultraviolet （UV）-B to induce the recurrent HSK. Corneal wholemounts were prepared at day 9 after modeling for the assessment of corneal neovascularization and nerve fiber distribution by immunofluorescence assay of CD31 and Ⅲ Tubulin antibodies. The areas of corneal neovascularization and scarring were measured with Image J software. The change rate of lesion was calculated and described as a ratio of lesion size at day 8 with day 0 after induction recurrence. Results The modeling success rate was over 80% ,and all infected mice showed latent period at day 45 after modeling. Corneal opacification was the most serious at day 7 after modeling and day 2 after recurrence,and the largest corneal neovascular area was seen at day 15 after modeling and at day 2 after recurrence, and the central cornea touch sensitivity was the worst at day 9 after initial infection. The mean corneal lesion area was 3. 348 mm2 in the ACV ＋bevacizumab-injected group, which was smaller than 3. 930 mm2 in the ACV-injected group （ Z = -2. 309,P = O. 021 ）. The central corneal sensitivity in the ACV＋bevacizumab-injected group was significantly higher than that in the normal saline-injected group （5.50±0.71 versus 0.50±1.41 ,Z=-2. 397 ,P=0. 029）. The increase rate of corneal lesion area in the ACV＋bevacizumab- injected group was evidently lower than that in the normal saline-injected group （ [ 167.10 ± 52.53 ] % versus [312.30±74. 18 ] % , Z = - 1. 992, P = 0. 046 ）. At the 7th day after modeling, the relative expressing levels of thymidine kinase （TK） and infected-cell protein-27 （ICP-27） mRNA in the corneal tissue and trigeminal ganglion were significantly increased at day 7 and reduced at day 45 after modeling,and the factors raised again at day 2 and retreated at day 7 after induction of recurrence. In addition, the expression of LAT mRNA peaked at day 45 after modeling and reduced gradually at day 2 after recurrence until a new increasing peak at day 7 after recurrence （ all at P〈0.01 ）. Immunofluorescence showed that compared with the normal saline-injected group, the corneal new vessels were lessened and corneal never fibers were increased in the ACV-injected group and ACV＋bevacizumab-injected group. Conclusions The combination of bevacizumab with ACV can inhibit corneal neovascularization and scarring in HSK mice,and bevacizumab exhibits a synergistic effect with ACV in management of HSK.