Miyata K, Murao M, Sawa M, Tanishima T
Department of Ophthalmology, University of Tokyo School of Medicine, Japan.
Jpn J Ophthalmol. 1990;34(3):267-74.
The roles of migration and mitosis in the in vitro corneal endothelial wound-healing process were studied using an immunohistochemical method. The in vitro wound-healing model was a cultured cell sheet prepared using bovine corneal endothelium, and the wound was made at the center of the cell sheet using a rotating silicon tip. Following the wounding, specimens were incubated with bromodeoxyuridine (BrdU) for 12 hours. After the fixation with 10% phosphate-buffered formalin, the cells incorporating BrdU were stained by the avidin biotin peroxidase complex method. In 12 hours after the wounding, no stained cells were observed. In 24 hours, cell migration had started and a few cells were stained; the average number of stained cells was 14.6 +/- 4.0 cells/mm2. In 48 hours, the number of stained cells increased. The maximum number of stained cells was observed between 48 and 60 hours after the wounding. The number of stained cells decreased rapidly after 72 hours when the wound was almost closed. Stained cells were most numerous in the area within 0.8 mm of the wound edge throughout the wound-healing. The endothelial wound-healing process in this model could be divided into four phases, latent, migration, concurrence of migration and mitosis, and inhibition phases. This in vitro wound-healing model could simulate the wound-healing process in vivo and will be useful for a quantitative evaluation of drug effects on the corneal endothelial wound-healing.
采用免疫组织化学方法研究了迁移和有丝分裂在体外角膜内皮伤口愈合过程中的作用。体外伤口愈合模型是使用牛角膜内皮制备的培养细胞片，使用旋转硅尖端在细胞片中心造成伤口。受伤后，将标本与溴脱氧尿苷（BrdU）孵育12小时。用10%磷酸盐缓冲福尔马林固定后，通过抗生物素蛋白生物素过氧化物酶复合物法对掺入BrdU的细胞进行染色。受伤后12小时，未观察到染色细胞。24小时时，细胞迁移开始，有少数细胞被染色；染色细胞的平均数量为14.6±4.0个细胞/mm²。48小时时，染色细胞数量增加。受伤后48至60小时观察到染色细胞数量最多。72小时后伤口几乎闭合时，染色细胞数量迅速减少。在整个伤口愈合过程中，伤口边缘0.8mm范围内的区域染色细胞最多。该模型中的内皮伤口愈合过程可分为四个阶段，即潜伏期、迁移期、迁移与有丝分裂并发期和抑制期。这种体外伤口愈合模型可以模拟体内伤口愈合过程，将有助于对药物对角膜内皮伤口愈合的作用进行定量评估。
