| 目的: 探讨氧自由基对准分子激光屈光性角膜切削术（photorefractive keratectomy, PRK）后凋亡机制介导的角膜创伤愈合反应的作用, 以及局部应用抗氧化剂维生素C(Vit C),维生素E(Vit E)对术后角膜的影响。 方法: 将28只兔分成3组，其中4只为正常对照;其余24只实验兔分成2组,每组12只，行双眼-5.0D PRK。一组术后左眼每日结膜下注射Vit C0.1g,右眼为对照；另一组术后左眼每日结膜下注射VitE25mg，右眼为对照。于术后1,3,7,14天测定角膜组织超氧化物歧酶(superoxide dismutase, SOD),丙二醛(malondialdehyde, MDA)及谷胱甘肽过氧化物酶(glutathioneperoxide, GPx)的变化，同时术后定期制作病理切片，检测角膜基质细胞数量,采用脱氧核苷酸末端转移酶介导的缺口末端标记法(terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling, TUNEL)检测角膜细胞凋亡,光镜观察凋亡细胞形态，定量统计比较凋亡水平差别。 结果: (1)PRK术后1,3天角膜SOD,GPx活性小于对照组(P<0.05),MDA的水平高于对照组(P<0.05)。局部应用抗氧化剂组其角膜SOD,GPx活性高于单纯激光组（P<0.05），MDA水平低于单纯激光组（P<0.05）。(2)角膜基质细胞凋亡在1-14天均与正常对照组有差别(P<0.01),在应用抗氧化剂组角膜基质细胞凋亡较单纯激光组减少（P<0.05）。(3)术后角膜基质细胞数增加,应用抗氧化剂组角膜基质细胞增生较单纯激光组减少（P<0.05）。 结论: 准分子激光屈光性角膜切削术后早期, 角膜存在着脂质过氧化形式介导的自由基性的组织损伤破坏,促进角膜细胞的凋亡；局部应用抗氧化剂VitC,VitE能早期减轻PRK术后炎性反应，降低角膜过氧化损伤，阻止术后细胞凋亡,减轻角膜基质反应性过度增生,降低术后屈光回退和haze形成。
Objective To investigate the effect of free oxygen radical in the rabbit cornea apoptosis-induced corneal wound healing respone after photorefractive keratectomy and the influence of topical antioxidants Vitamin C, Vitamin E on cornea tissue having undergone PRK.
Methods 28 rabbits were divided 3 groups, 4 rabbits served as controls and the others served as experimental rabbits which were divided 2 groups, each consisting of 12 animals. They were underwent bilateral photorefractive keratectomy with 193nm argon fluoride excimer laser to correct 5 diopters of myopia. Following treatment, one group the left eye of each rabbit was subconjunctival injection with Vitamin C 0.1 every day. The right eye served as control. The other group the left eye of each rabbit was subconjunctival injection with Vitamin E 25mg every day. The right eye served as control. Rabbits were killed at 1,3,7 and 14 days respectively, then measured the activity of corneal superoxide dismutase(SOD) with xanthine oxidase method and glutathioneperoxide(GPx) with modified glutathione, the level of corneal malondialdehyde(MDA) with barbituric acid reaction. At the same time, corneas were prepared for H.E.staining to observe histopathological changes and the quantities of corneal stroma cells after PRK. Corneal cell apoptosis was evaluated by terminal deoxyribonucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling (TUNEL) to detect DNA fragmentation.
Results (1) It was found that significant decrease in SOD, GPx activity (P<0.05) and increase in MDA level (P<0.05) compared with the control following 1,3 days after PRK. But the corneal SOD, GPx activity in effect of antioxidants group is significant higher than that of PRK group (P<0.05). The level of MDA in effect of antioxidants group is significant lower than that of PRK group (P<0.05). (2) Apoptosis was detected remarkably in anterior keratocytes from 1 day to 14 days (P<0.01). The level of keratocyte apoptosis in effect of antioxidants group less then PRK group (P<0.05). (3) The number of keratocytes were increased after operation. The level of proliferation in effect of antioxidants group less then PRK group (P<0.05).
Conclusions The presence of free oxygen radical mediated tissue damage in the cornea with lipid peroxidation following excimer laser keratectomy. They may contribute to an increase apoptosis in cornea after PRK. The topical application of antioxidants Vitamin C or Vitamin E can decrease corneal oxygen radical tissue damage after excimer laser keratectomy, inhibiting keratocyte apoptosis, preventing the over-proliferation and all of them may reduce postoperative corneal haze and regression.
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