［摘要］目的:评价实验性家兔不同屈光度PRK术后角膜创面愈合的过程及皮质类固醇对愈合的影响。方法:应用SVS APEX PLUS型准分子激光治疗系统对6只家兔双眼进行PRK术，设定右眼-4.00D，左眼-8.00D。随机分为FLM点眼组和CM点眼组，每组3只家兔，FLM点眼组双眼滴皮质类固醇激素眼液（0.1％ fluorometholone，FLM，美国）；CM点眼组双眼滴0.25％氯霉素眼液。分别于术后3天、30天、100天两组随机选取1只家兔，猝死摘除双眼球，取其角膜。每只角膜分为两半，一半角膜电镜下观察其超微结构；另一半OCT包埋后冰冻切片，用于免疫组化研究，检测Ⅲ型胶原(Ⅲ-C)和连接蛋白(FN)，免疫组化切片后剩余部分，石蜡包埋，行HE染色，光镜下组织学观察。结果:术后100天，除1只家兔角膜haze为1级外，余家兔角膜haze为0级。术后100天，角膜上皮细胞基本恢复正常。上皮细胞界限分明，上皮明亮细胞较多，但微绒毛、微皱褶相对减少。CM组Ⅲ-C和FN的表达较FLM组明显；左眼(-8.00D)Ⅲ-C和FN的表达较右眼(-4.00D)明显。结论:PRK术后100天角膜组织结构基本正常，但仍有组织薄弱处和特殊性改变。角膜基质层切削深度可能是影响PRK术后haze发生的一个因素。PRK术后家兔角膜上皮微绒毛、微皱褶相对减少，可能是临床上PRK术后部分患者近期角膜干燥的原因之一。

［中图分类号］R779.63［文献标识码］A［文章编号］1008-1801(2000)02-0094-05

A histopathologic immunohistochemical and ultrastructural study of rabbit cornea after eximer laser photorefractive keratectomy

ZHANG Hua

（Xi'an Medical University, Xi'an 710061）

HU Hai-tao

（Xi'an Medical University, Xi'an 710061）

REN Hui-min

（Xi'an Medical University, Xi'an 710061）

Abstract：Objective：To evaluate the corneal healing of unequal dioptric response using histopathological, immunohistochemical and ultrastructural techniques after PRK with SVS APEX PLUS excimer laser. The effects of corticosteroid on healing were also studied.Methods:PRK was performed on both eyes of 6 white rabbits (12 eyes). PRK was performed on the right eye of the rabbit to attempt a correction of -4.00 diopters, and PRK was performed on the left eye of the rabbit to attempt a correction of -8.00 diopters. The subjects were randomly divided into two groups: the FLM group (3 rabbits, 6 eyes) and the CM group (3 rabbits, 6 eyes). Corticosteroid was given to the eyes of the FLM group, but not the CM group. The eyes were re-examined at 10 days, 30 days and 100 days. The eyes of one rabbit from each group were randomly selected and enucleated. Half of each cornea was prepared for electron microscope study (SEM and TEM); the other half was embedded in an OCT compound for immunohistochemical study to examine Ⅲ-C and FN.Results:All eyes reepithelialized within 3 days after PRK. Subepithelial corneal haze was observed at 15 days but was not dominant at 30 or 60 days. At 100 days post-operatively, 11 eyes with corneal haze had grade 0 or 0.5. Only one eye (the left eye from the CM group) showed a denser haze (grade 1) at 100 days. At 3 days post-operatively one or two layers of corneal epithelial cells covered the ablation zone. At 30 days after PRK, the epithelial cells showed hyperplastic changes. The cells were larger and increased to 7 or 8 layers of cells from the normal 5 or 6 layers. At 100 days after PRK, the epithelium was clear and brighter. Microplicae and microvilli were less evident than before. There was significantly more expression of Ⅲ-C and FN in the CM group than in the FLM group.Conclusion:We found that despite recovery of a continuous and smooth epithelial layer and near-normal corneal tissues 100 days after PRK, abnormalities of both epithelium and superficial stroma could be detected in the area of ablation. The ablation depth in the stroma can influence the appearance of corneal haze after PRK. The microplicae and microvilli of rabbit corneal epithelium was reduced after PRK, which may be one of the reasons for the sensation of dry eye in some patients.

Key words：excimer laser; photorefractive keratectomy (PRK); cornea/pathology; cornea/ultrastructure; wound healing; type Ⅲ collagen (Ⅲ-C); fibronectin (FN); rabbits准分子激光角膜切削术(Excimer laser photorefractive keratectomy,PRK)的问世，开创了角膜屈光手术的新时代^［1］。PRK是在去除角膜上皮后，用准分子激光对角膜中央的前弹力层和前基质层进行切削，使角膜中央的前表面相对变平，从而达到矫正近视的目的。由于它与传统的角膜屈光手术在原理和手术方法上都有许多不同之处^［2～4］，因此，有必要对PRK术后创面愈合过程及其控制作进一步的研究。本研究利用电镜、光镜及免疫组化技术，来研究家兔不同屈光度PRK术后角膜愈合过程中的组织病理学改变及术后用药对这一过程产生的影响。

1材料和方法

1.1实验动物健康白色家兔6只，体重2.0～2.5kg，第四军医大学实验动物饲养中心提供，雌雄兼用。

1.2主要仪器与试剂

1.2.1主要仪器准分子激光系统：SVS APEX PLUS型，美国森美公司。H-600透射式电子显微镜，日本。KyKy-2000扫描电子显微镜：中国科学院科学仪器厂。

1.2.2主要试剂包埋剂：D-6907，德国。一抗：鼠抗人Ⅲ-C单克隆抗体（上海生物制品有限公司产品），二抗：鼠抗人FN单克隆抗体(sigma 公司产品)。0.1％艾氟龙(fluorometholone)：美国眼力健公司。

1.3实验方法

1.3.1手术方法采用SVS APEX PLUS型准分子激光治疗系统对6只家兔双眼进行PRK术，设定右眼-4.00D，左眼-8.00D。按设定的屈光度切削，-4.00D采用单中心一次性切削，-8.00D采用多区切削(Stepped multi-zone PRK)，切削方式同Zhang等^［4］已报道的方法。术后3天、30天和100天进行复查，内容包括：角膜上皮的再生，角膜haze。术后3天内术眼滴0.5％四环素可的松眼膏，每日三次。随机分为FML点眼组和CM点眼组，每组3只家兔，FML点眼组双眼滴皮质类固醇激素眼液(0.1%FML)；第一个月每日4次，第二个月每日3次，第三个月每日2次。CM点眼组双眼滴0.25％氯霉素眼液。分别于术后3天、30天、100天两组随机各选取1只家兔，猝死摘除双眼球，取其角膜。每只角膜分为两半，一半角膜电镜（透射电镜和扫描电镜）下观察其超微结构(1/4行SEM，1/4行TEM)；另一半OCT包埋后冰冻切片，用于免疫组化研究，检测Ⅲ-C和FN，免疫组化切片后剩余部分，石蜡包埋，行HE染色，光镜下行组织学观察。

1.3.2免疫组化冰冻切片制作常规取材，包埋剂包埋、冰冻，切片厚6μm，放入pH7.6 TBS缓冲液中冲洗；加入10％正常羊血清30分钟，室温，取掉多余血清；加入1：50稀释一抗过夜，TBS冲洗三次；加入二抗30分种，温度37℃，TBS冲洗三次；加入0.1％H₂O₂，甲醇中室温保存30分钟，TBS冲洗三次；加入复合物30分种，温度37℃，TBS冲洗三次；DAB加入10ml TBS中，加入20mlH₂O₂显色，镜下控制；终止显色后，苏木素复染核；常规分化脱水透明，树胶封片。

1.3.3电镜标本制作2.5％戊二醛前固定2h(4℃)后，1％四氧化锇后固定1h，乙醇系列脱水，Epon812包埋，超薄切片机切片约50nm，铅、铀染色，透射电镜观察拍照。扫描电镜样品经2.5％戊二醛固定后，在液氮内垂直断裂，1％四氧化锇后固定，乙醇系列脱水，醋酸异戊酯置换，临界点干燥，喷金后用扫描电镜观察拍照。

1.4角膜haze临床分级参照Snibson等^［5］制定的标准。

2结果

2.1裂隙灯观察所有家兔角膜上皮均在PRK术后第3天愈合，术后15天开始出现不同程度的角膜haze，30～60天时最明显，术后100天，除1只家兔角膜haze为1级外，余家兔角膜haze为0级。

2.2光镜观察术后3天，角膜上皮细胞1层或2层覆盖切削区。术后30天，可见角膜上皮细胞增生，体积增大，细胞由正常的5层或6层增加到7层或8层。术后100天，角膜上皮细胞基本恢复正常。见图1～图4。

2.3透射电镜观察术后3天，角膜上皮细胞1层或2层覆盖切削区，角膜前基质胶原纤维排列紊乱，深层基质胶原纤维排列较整齐，后弹力层和内皮细胞结构未见明显改变。术后30天，可见角膜上皮细 胞增生，体积增大，细胞由正常的5层或6层增加到7层或8层<