兔角膜基质飞秒激光扫描后Ki-67、转化生长因子β2及平滑肌肌动蛋白的表达

The expression of Ki-67, TGF-β2 and α-SMA after the femtosecond laser-assisted intrastromal scanning in rabbit cornea

目的研究角膜基质飞秒激光扫描后Ki-67、转化生长因子β2及平滑肌肌动蛋白（α-SMA）的表达情况，探讨角膜基质内扫描后角膜创伤愈合反应机制。方法20只纯种新西兰大白兔随机编号，35只眼用60kHzIntralase飞秒激光进行角膜基质内扫描（其中5只兔只做右眼），未手术的5只左眼作为正常对照组。激光参数为：点／线间距10μm，扫描直径8．5mm，扫描能量1．3μJ，扫描深度为135μm，不进行角膜边缘切削。术后在30min、1h,2h、1个月和3个月用裂隙灯显微镜观察角膜的透明程度。分别在术后1d．3d、1周、1个月和3个月各取6只术眼的角膜组织，做免疫印迹法蛋白定量检查，各时间点取1只术眼做免疫组织化学检查，分别观察Ki-67、TGF-β2及α-SMA的表达情况。用两组完全随机化设计资料均数的t检验进行统计学处理。结果免疫印迹法蛋白定量及免疫组织化学检查显示：Ki-67在基质扫描后第1天开始表达增加（0．0670±0．0008），扫描后3d达最高峰（0．6923±0．0051），随着时间推移其表达逐渐减少，但直至术后3个月和对照组相比差异均有统计学意义（t=24．12，57．22，43．26，39．78，18．35；P〈0．05）。TGF-β2（t=0．933，0．856，0．934，0．970，1．132）及OL-SMA（t=1．126，1．235，0．993，1．175，1．211）在术后各时间点的表达和对照组相比差异均无统计学意义（P〉0．05）。结论角膜基质内飞秒激光扫描后Ki-67表达增加，角膜基质细胞活化、增殖；TGF—β2被完整的角膜上皮所屏障，活化和增生的角膜基质细胞没有向肌成纤维母细胞转化，损伤修复愈合过程中无haze形成。

Objective To study how the cornea would express Ki-67, TGF-β2 and α-SMA and how the cornea would heal when it is scanned intrastromally. Methods Twenty New Zealand white rabbits were randomly numbered and used for the experiments. The 60 kHz Intralase femtosecond laser delivery system was used to scan 35 rabbit corneas （of these corneas, 5 right corneas were from 5 rabbits）. The unoperated 5 left eyes were used as control. The laser settings were： spot/line separation, 10 μm; diameter, 8.5 mm; energy for scanning the stroma, 1.3 p J; scanning depth, 135 μm, no edge cuts were performed. Slit lamp was used to observe the cornea at 0. 5 h, 1 h, 2 h, and 3 months after surgery. On day 1, 3, 7, 30, 90 after surgery, 6 and 1 scanned corneas at each time point were taken for Western blot and immunocytochemical detection respectively to detect the expression of TNF-α, Ki-67, TGF-132 and α-SMA. Results Microbubbles were found in corneal stroma after surgery. The number of microbubbles tended to decrease significantly with time. At 2 h, the cornea turned to be transparent again. During 3-month follow- up, the cornea was always transparent and no haze appeared. Western blot analysis and immunocytochemical detection demonstrated that the expression of Ki-67 began to increase at day 1 （ 0. 0670 ＋ 0. 0008 ） and reached the peak at day 3 after surgery（0. 6923 ＋0. 005）. While there were significant difference with the control group at day 90 after surgery （ t = 24. 12,57.22,43.26, 39. 78, 18.35 ; P 〈 0. 05 ）, a gradual decrease in expression of ki-67 could be detected from day 3 to 90. There were no significant change in the expression of TGF-132 （ t = 0. 933,0. 856,0. 934,0. 970, 1. 132 ） and α-SMA （ t = 1. 126, 1. 235,0. 993, 1. 175,1.211 ） at all time points after surgery （P 〉 0. 05 ）. Conclusions After intrastromal scanning, the expression of Ki-67 increases and the keratocyte is activated and proliferated. However, due to no epithelium injured and TGF-β2 confined to the epithelium, the activated and proliferated keratocytes could not be transformed into myofibroblasts.