Petroll W M, Jester J V, Bean J, Cavanagh H D
Department of Ophthalmology, University of Texas Southwestern Medical Center at Dallas, 75235-9057, USA.
Cornea. 1999 Jan;18(1):98-108.
To assess the efficacy of labeling actively cycling corneal endothelial cells by using a monoclonal antibody to the Ki67 antigen (MIB-1) and to determine what changes in f-actin and ZO-1 organization are associated with entry into the cell cycle during wound healing under different culture conditions.
Three corneal buttons (6 mm diameter) were punched from each cornea of 15 cats. After a mechanical scrape injury (2 mm diameter) was made, buttons were cultured for 24, 48, or 72 h in serum-free media (SFM), SFM plus 10% fetal calf serum, or SFM plus basic fibroblast growth factor (bFGF). Buttons were single and double labeled by using phalloidin, anti-ZO-1, and MIB-1. Counts of Ki67-positive cells were used to determine the number of actively cycling endothelial cells.
After culture in SFM, wounds healed by cell spreading with maintenance of normal apical f-actin and ZO-1 organization; Ki67-positive cells were detected near the leading edge in some areas. A significant increase in the number of cycling cells was measured after 48 h of culture in bFGF as compared with SFM (p<0.05); serum increased the number of cycling cells more than both SFM and bFGF (p<0.05). In all cases, positive MIB-1 staining was not observed until 48 h after injury, was limited to cells actively spreading over the wound area, and was diminished after wound closure (72 h). Double labeling demonstrated that endothelial cells exhibited a fibroblastic phenotype in some central areas of cell proliferation after culture in serum or bFGF, but, in general, apical cell border-associated f-actin and ZO-1 organization was partially maintained in most Ki67-positive cells.
The data suggest that spreading corneal endothelial cells are capable of proliferating and can respond to growth factors, but that dedifferentiation or fibroblastic transformation is not required before entry into the cell cycle. Overall, the MIB-1 antibody appears to be ideally suited to the study of corneal endothelial proliferation during wound healing.
评估使用抗Ki67抗原单克隆抗体（MIB-1）标记活跃循环的角膜内皮细胞的效果，并确定在不同培养条件下伤口愈合过程中，f-肌动蛋白和紧密连接蛋白1（ZO-1）组织的哪些变化与进入细胞周期相关。
从15只猫的每只角膜上取下三个角膜纽扣（直径6毫米）。在造成机械刮伤（直径2毫米）后，将纽扣在无血清培养基（SFM）、SFM加10%胎牛血清或SFM加碱性成纤维细胞生长因子（bFGF）中培养24、48或72小时。使用鬼笔环肽、抗ZO-1和MIB-1对纽扣进行单标记和双标记。通过计数Ki67阳性细胞来确定活跃循环的内皮细胞数量。
在SFM中培养后，伤口通过细胞铺展愈合，顶端f-肌动蛋白和ZO-1组织保持正常；在某些区域的前沿附近检测到Ki67阳性细胞。与SFM相比，在bFGF中培养48小时后，循环细胞数量显著增加（p<0.05）；血清增加的循环细胞数量比SFM和bFGF都多（p<0.05）。在所有情况下，直到受伤后48小时才观察到MIB-1阳性染色，仅限于在伤口区域积极铺展的细胞，并且在伤口闭合后（72小时）减少。双标记显示，在血清或bFGF中培养后，内皮细胞在细胞增殖的一些中央区域表现出成纤维细胞表型，但总体而言，大多数Ki67阳性细胞中顶端细胞边界相关的f-肌动蛋白和ZO-1组织部分保持。
数据表明，铺展的角膜内皮细胞能够增殖并能对生长因子作出反应，但进入细胞周期前不需要去分化或成纤维细胞转化。总体而言，MIB-1抗体似乎非常适合用于研究伤口愈合过程中角膜内皮细胞的增殖。
