表层切削术后早期应用碱性成纤维细胞生长因子对兔角膜上皮下雾状混浊的作用及其机制

Effects of early applying basic fibroblast growth factor on corneal haze after surface ablation surgery and its mechanism

目的探讨兔角膜表层切削术后早期应用碱性成纤维细胞生长因子(bFGF)对角膜上皮下雾状混浊(haze)的作用及其机制。方法选取60只健康普通级新西兰大白兔,均行右眼准分子激光角膜切削术(PRK),术后采用随机数字表法将实验眼分为PRK+生理盐水组、PRK+bFGF组和单纯PRK组,每组20眼,术后分别给予生理盐水和bFGF点眼,每日3次,每次1滴,单纯PRK组未行任何处理,药物连续应用至动物处死,另取8只兔为空白对照组。各手术组分别于术后7d和28d各取10只兔,空白对照组取4只兔,通过眼前节照相、眼前节光相干断层扫描成像(AS-OCT)记录角膜上皮愈合及haze情况,并行Fantes分级;采用苏木素-伊红染色法观察兔角膜组织病理学变化;采用免疫组织化学法检测兔角膜组织中转化生长因子-β1(TGF-β1)、α-平滑肌肌动蛋白(α-SMA)、基质金属蛋白酶-2(MMP-2)的表达。结果各手术组角膜上皮愈合时间比较差异无统计学意义(F=0.57,P=0.57)。各组兔眼术后不同时间点haze分级总体比较差异均有统计学意义(F组别=41.736,P<0.01;F时间=129.445,P<0.01);术后28d,PRK+bFGF组haze分级明显高于PRK+生理盐水组和单纯PRK组,差异均有统计学意义(均P<0.05)。AS-OCT显示,空白对照组角膜上皮表面反射条带连续、光滑,上皮细胞与基质层排列紧密;术后7d各组角膜上皮表面反射条带连续、光滑,与浅基质层连接欠紧密,浅基质层反射增强,组织病理学检查发现术区角膜上皮细胞和基质层细胞增生,基质层胶原纤维排列紊乱,其中以PRK+bFGF组最明显;术后28d,上述表现明显增强。免疫组织化学染色结果显示,空白对照组角膜组织中仅见少量MMP-2表达;术后7d各手术组角膜组织中TGF-β1、α-SMA、MMP-2均呈阳性表达,PRK+bFGF组角膜组织中阳性染色最强;术后28d,TGF-β1、α-SMA和MMP-2的表达较术后7d明显增加,差异均有统计学意义(均P<0.05)。结论表层切削术后早期应用bFGF可促进haze形成,其主要机制与bFGF促进角膜上皮细胞增生,影响浅基质层胶原排列,导致haze相关因子TGF-β1、α-SMA和MMP-2表达增加有关。

Objective To investigate the effects of early applying of basic fibroblast growth factor(bFGF)on corneal haze formation after surface ablation surgery in rabbits.Methods The right eyes of 60 healthy New Zealand white rabbits received photorefractive keratectomy(PRK)and were randomized into PRK+normal saline group,PRK+bFGF group and simple PRK group,with 20 rabbits in each group.Normal saline solution and bFGF were topically administered according to grouping,respectively,3 times per day,1 drop for each time until the sacrifice of the animals,and no drug was used in the PRK group.Another 8 normal rabbits were served as blank control group.The corneal healing response and haze formation were evaluated by anterior segment photography and anterior segment optical coherence tomography(AS-OCT)and graded based on Fantes criteria.Corneal histopathology was examined by hematoxylin-eosin staining.Immunohistochemistry was used to detect the expression of transforming growth factor-β1(TGF-β1),α-smooth muscle actin(α-SMA)and matrix metalloproteinase-2(MMP-2)in cornea.This study protocol was approved by the Experimental Animal Ethic Committee of Affiliated Hospital of Binzhou Medical University(20180209-03).The use and care of the animals complied with the Statement of ARVO.Results The corneal epithelium was completely healed in 3-4 days following surgery and there was not significantly different in healing time among the three groups.(F=0.57,P=0.57).The haze grading was significantly different among different groups at different time points(Fgroup=41.736,P<0.01;F time=129.445,P<0.01)and showed the highest score in the PRK+bFGF group on the 28th day after operation.On the 7th day after surgery,AS-OCT image showed that the surface reflection of corneal epithelium was continuous and smooth and corneal epithelium was not tightly attached to the superficial stromal layer;the reflection of the superficial stromal layer was enhanced in all the operation groups.The proliferation of corneal epithelial cells and superficial stromal layer in the operation area were seen under the optical microscope,and the arrangement of collagen fibers in the stromal layer was disordered with the most obvious changes in the PRK+bFGF group in comparison with the PRK+normal saline group and the simple PRK group,and these findings became worse on postoperative 28 days.The corneal epithelial surface reflection in the blank control group was continuous and smooth.Immunohistochemistry showed that a few MMP-2 positive cells were seen in the blank control group.TGF-β1,α-SMA and MMP-2 proteins were positively expressed in the corneas 7 days after surgery in the three groups,and their expressions were the most obvious in the PRK+bFGF in comparison with the PRK+normal saline group and the PRK group and were enhanced 28 days after operation,showing statistically differences(all atP<0.05).Conclusions Early application of bFGF following surface ablation surgery promotes the proliferation of corneal epithelial cells and irregular arrangement of collagen in the superficial stromal layer,which is associated with the expressions of haze-related factors TGF-β1,α-SMA and MMP-2 in corneas.