方法：新西兰大白兔64只行右眼PRK手术后，根据术后局部用药不同，随机分为4组：单纯手术组、14μmol/L二甲基亚砜组(DMSO)、50μmol/L雷帕霉素组、100μmol/L雷帕霉素组，每组16只。根据分组情况，术后2h开始给予上述滴眼液治疗，每天3次，1次1滴，持续7d，另选取16只兔为正常对照组。术后每天用手持裂隙灯显微镜照相系统，观察兔眼术后炎症反应及角膜上皮愈合情况。裂隙灯显微镜照相系统采集PRK术后1、4wk时各组兔眼haze的形成情况。各组分别于术后1、4wk采用空气栓塞法处死8只兔子，摘取角膜组织，冻存备用。免疫组化检测转化生长因子-β1(TGF-β1)、α-平滑肌肌动蛋白(α-SMA)、基质金属蛋白酶-2(MMP-2)的表达水平。Real Time-PCR技术检测自噬因子-5(ATG5)、自噬因子-12(ATG12)、B淋巴细胞瘤基因-2(Bcl-2)、半胱氨酸天冬氨酸蛋白酶-3(Caspase3)基因的相对表达量。

结果：PRK术后兔眼角膜上皮均在3～4d完全愈合，各组兔眼术后角膜上皮愈合时间比较无差异(F=0.745，P=0.530)。在观察期间，各组术后haze均于4wk时最明显，单纯手术组和14μmol/L DMSO组haze症状均较严重，其次为50μmol/L雷帕霉素组，100μmol/L雷帕霉素组haze症状较其他组明显减轻，各组兔眼术后不同时间点haze分级比较有差异(均P<0.05)。免疫组化显示，术后1、4wk，单纯手术组和14μmol/L DMSO组TGF-β1、MMP-2和α-SMA的表达均较强，其次为50μmol/L雷帕霉素组，100μmol/L雷帕霉素组较其他组表达明显减弱(均P<0.05)。PCR法检测显示，术后1、4wk，50、100μmol/L雷帕霉素组ATG5、ATG12和Bcl-2 mRNA相对表达量较单纯手术组和14μmol/L DMSO组明显升高(均P<0.05)； 50、100μmol/L雷帕霉素组Caspase3 mRNA相对表达量较单纯手术组和14μmol/L DMSO组明显降低(均P<0.05)。

结论：雷帕霉素可以增强自噬水平，抑制凋亡水平，从而减轻兔眼PRK术后haze的形成。

METHODS: Totally 64 New Zealand white rabbits were randomly divided into 4 groups according to different postoperative medication after PRK operation, including simple PRK group, 14μmol/L DMSO group, 50μmol/L rapamycin group and 100μmol/L rapamycin group. According to the group situation, two hours after the operation, eye drops were given, 3 times a day for 7d. Another 16 rabbits were selected as normal control group. The postoperative inflammatory response and corneal epithelial healing were observed with slit-lamp microscope every day. Haze formation of each group at 1 and 4wk after PRK was collected by slit-lamp microscopy system. Eight rabbits in each group were killed by air embolization 1 and 4wk after PRK, and corneal tissue was extracted and frozen for later use. Immunohistochemistry was used to detect the expression levels of transforming growth factor-β1(TGF-β1), α-smooth muscle actin(α-SMA), and matrix metalloproteinase-2(MMP-2). Real-time PCR was used to detect the relative expression levels of autophage-5(ATG5), autophage-12(ATG12), B lymphocytoma gene-2(Bcl-2)and cysteine aspartic proteinase-3(Caspase3)genes.

RESULTS: Corneal epithelium of all operative rabbits healed completely at 3-4d and no significant difference in healing time between the groups after operation(F=0.745, P=0.530). During the observation period, haze was the most obvious at 4wk after operation in all groups. The haze symptoms were more serious in the simple operation group and the 14μmol/L DMSO group, followed by the 50μmol/L rapamycin group. The haze symptoms in the 100μmol/L rapamycin group were significantly relieved than those in other groups. There was no significant difference in the haze grading with different time points after operation among all groups(all P<0.05). Immunohistochemistry showed that the expression of TGF-β1, MMP-2 and α-SMA was stronger in the operation group and 14μmol/L DMSO group, followed by 50μmol/L rapamycin group, and weakest in 100μmol/L rapamycin group than other groups at 1 and 4wk after operation(all P<0.05). The results of PCR showed that the relative expression of ATG5, ATG12 and Bcl-2 mRNA in 50μmol/L rapamycin group and 100μmol/L rapamycin group were significantly higher than those in simple operation group and 14μmol/L DMSO group at 1 and 4wk after operation(all P<0.05); The relative expression of Caspase3 mRNA in 50μmol/L rapamycin group and 100μmol/L rapamycin group was significantly lower than that in simple operation group and 14μmol/L DMSO group(all P<0.05).

CONCLUSION: Rapamycin can enhance autophagy level and inhibit apoptosis level, thus reducing haze formation after PRK in rabbits.