目的 探讨羊膜提取液(AE)对兔PRK术后haze的影响及机制.方法 实验研究.36只新西兰白兔(72只眼)用随机数字表法随机分为3个组(实验组、激素组与空白组),构建PRK动物模型,术后实验组滴用AE,激素组滴用0.1％磷酸地塞米松眼液,空白组滴用不含AE的赋形剂眼液.用裂隙灯眼前节照相记录haze的形态,荧光素钠染色观察角膜上皮的修复,TUNEL法观察角膜基质细胞的凋亡,免疫荧光法观察角膜基质细胞α-SMA的表达.用Kruskal-Wallis H检验对haze 分级评分进行统计学分析,用方差分析及LSD-t检验进行多组间均数的比较,用Spearman等级相关分析对haze形成与TUNEL阳性细胞及α-SMA表达量进行相关性分析.结果 72只兔眼角膜上皮均在PRK术后6d内完全修复,实验组、激素组及空白组角膜上皮平均修复时间分别为(4.12±0.62)、(5.25±0.78)及(4.96±0.73)d,组间比较差异有统计学意义(F=14.144,P＜0.01).3个组术后1周均开始出现不同程度的haze,3～4周时尤其明显,术后8周减轻.实验组角膜haze程度与激素组和空白组比较,术后1周(H=3.995,12.77;P＜0.05)、4周(H=4.468,9.003;P＜0.01)及8周(H=4.397,5.744;P＜0.05)均较低.术后1周实验组、激素组及空白组TUNEL阳性细胞数分别为(2.2500 ±0.3750)、(4.5000±0.7500)和(7.1250 ±0.9063)个/高倍视野,实验组与空白组和激素组比较差异均有统计学意义(t =4.26,8.13;P＜0.01);术后4周空白组仍有表达,但实验组、激素组未见明显阳性细胞表达;术后8周3个组均未见明显TUNEL阳性细胞表达.术后1～8周,α-SMA在空白组、激素组的角膜基质浅层均有阳性表达,且2个组术后4周阳性细胞数目较1周增高,术后8周较4周的阳性细胞数目减少.术后1～8周,α-SMA在实验组均为阴性表达.实验组与激素组、空白组相比,术后1周(t=28.62,36.55;P＜0.01)、4周(t=30.40,35.96;P＜0.01)、8周(t=34.02,38.32;P＜0.01)差异均有统计学意义.Spearman等级相关分析,显示haze形成与TUNEL阳性细胞表达量(r =0.881,P＜0.01)及α-SMA表达量(r=0.710,P＜0.01)呈正相关.结论 AE可有效抑制PRK术后haze的形成,其机制可能与AE能够降低角膜细胞的凋亡,减少肌成纤维细胞的生成与聚集,从而减少胶原与瘢痕形成有关.%Objective To investigate the effects and mechanism of amniotic extraction on corneal healing after photorefractive keratectomy (PRK).Methods Experimental Study.Thirty-six New Zealand rabbit corneas were performed with PRK models (-10 diopters,6.5 mm diameter).According to random number table,all eyes were divided into three groups,including treated with amniotic extraction,0.1％dexamethasone and excipient respectively after operation.Clinical and histopathologic examinations were taken by slit-lamp microscope and fight microscope.Corneal epithelium reparation was observed by fluorescent staining.Corneal stroma cell apoptosis was evaluated by terminal deoxyribonucleotidy transferasemediated deoxynuridine triphophate nick end labeling (TUNEL) assay.Myofibroblast generation was evaluated by immunofluorescence checking the expression of alpha-smooth muscle actin (α-SMA).The number of TUNEL and α-SMA positive cells was analyzed to explore the effects on corneal haze.The haze grading was compared between groups using Kruskal-Wallis H test.Mean values for each experiment were compared between groups using a one-way analysis of variance and LSD-t test.Spearman rank analysis was used to evaluate the correlation between the haze grading and the expression of TUNEL positive cells and α-SMA.Results The corneas of seventy-two eyes reepithelialized in 6 days after operation.The average epithelium repair time of the AE group was (4.12 ± 0.62) d,the dexamethasone group was (5.25 ±0.78) d,and the excipient group was (4.96 ± 0.73) d.The progression of reepithelialization wassignificantly faster in the AE group than the other two groups (F =14.144,P ＜0.01).The haze appeared in the first week after the PRK in all three groups,increased after 3-4 weeks,and relieved after 8 weeks.The degree of haze was significantly lower in the AE group than the other two groups in the first week (Vs.dexamethasone group,H =3.995,P ＜ 0.05 ; vs.excipient group,H =12.77,P ＜ 0.01),in the 4th week (Vs.dexamethasone group,H =4.468,P ＜ 0.05 ; vs.excipient group,H =9.003,P ＜ 0.01),and 8th week (Vs dexamethasone group,H =4.397,P ＜ 0.05 ; vs.excipient group,H =5.744,P ＜ 0.05) after PRK.The TUNEL-positive cells appeared in the central anterior stroma at the first week after surgery.And the number of TUNEL-positive cells in the AE group was (2.2500 ± 0.3750) cells/HP,the dexamethasone group was (4.5000 ±0.7500) cells/HP,and the excipient group was (7.1250 ±0.9063) cells/HP.The number of TUNEL-positive cells in AE group was less than those in the other two groups (Vs.dexamethasone group,t =4.26,P ＜0.01 ; vs.excipient group,t =8.13,P ＜0.01).The TUNEL-positive cells were only found in the excipient group (2.8750 ± 0.6563)/HP in 4th week after operation.It was significantly different between the dexamethasone group and the excipient group (t =9.01,P ＜ 0.01).There were no significant TUNEL-positive cells in 8th weeks in all three groups.α-SMA-positive cells started to appear apparently at the first week after surgery in the dexamethasone and excipient groups,and the peaks appeared at the 4th week after treatments,and there were still a lot of α-SMA-positive cells in corneal stroma at the 8th week after operation in both groups.On the contrary,there were no significant α-SMA-positive cells in the AE group all the time after surgery.The statistical significant difference can be found between the AE group vs.the dexamethasone and excipient groups in the first week (t =28.62,36.55 ;P ＜0.01),in the4th week (t=30.40,35.96; P ＜0.01),and in the 8th week (t =34.02,38.32; P＜0.01).Spearman rank analysis demonstrated that the formation of haze was proportional to the expression of TUNEL positive cells (r =0.881,P ＜ 0.01) and α-SMA (r =0.710,P ＜ 0.01).Conclusion Amniotic extraction can reduce the formation of haze,which was more effective than 0.1％ dexamethasone.It might release certain factors which were transported into corneal matrix,then affected the healing of epithelial cell by interacting with the corneal cell factors,reducing the cell apoptosis,corneal wound healing response and rebuilding the corneal matrix with less myofibroblast,collagen and scar and finally reduce the formation of haze.
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