Zieske J D, Bukusoglu G, Yankauckas M A
Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114.
Invest Ophthalmol Vis Sci. 1992 Jan;33(1):143-52.
Corneal epithelial stem cells are thought to be localized in the basal cell layer of the limbus. We developed a monoclonal antibody designated 4G10.3 that immunolocalized to limbal basal cells in rat corneas. Western blot analysis demonstrated that 4G10.3 reacted with a single band of 50,000 molecular weight in rat and rabbit corneal epithelium and 48,000-49,000 in human epithelium. Following extraction of corneal epithelium in 20 mM Tris-HCl (pH 6.8) and ultracentrifugation at 100,000 x g, the 50-kD protein was detected in the soluble fraction. 4G10.3 also was used to examine the response of the limbal basal cells to epithelial debridement and thermal burn wounds in the rat. In unwounded control corneas, 40.8 +/- 12.0 (mean +/- standard deviation) cell per limbal area bound 4G10.3. Following a 3 mm debridement wound, the number of cells binding 4G10.3 increased to 77.0 +/- 16.9 two days post-injury and returned to control levels by three days. Following thermal burn, 36.2 +/- 11.2, 68.8 +/- 15.8, 85.4 +/- 15.4, 104.6 +/- 13.8, and 88.0 +/- 40.2 cells per limbal area bound 4G10.3 18 hours, and 1, 2, 3, and 7 days post-injury, respectively. The 50 kD protein and 4G10.3 antibody provided a biochemical and immunological marker of limbal basal cells, hypothesized to be corneal epithelial stem cells.
角膜上皮干细胞被认为定位于角膜缘的基底细胞层。我们研发了一种名为4G10.3的单克隆抗体，它能在大鼠角膜的角膜缘基底细胞中进行免疫定位。蛋白质印迹分析表明，4G10.3与大鼠和兔角膜上皮中一条分子量为50,000的单条带发生反应，在人上皮中则与分子量为48,000 - 49,000的条带发生反应。在20 mM Tris - HCl（pH 6.8）中提取角膜上皮并以100,000 x g进行超速离心后，在可溶部分检测到了50 - kD蛋白。4G10.3还被用于检测大鼠角膜缘基底细胞对上皮清创和热烧伤创面的反应。在未受伤的对照角膜中，每角膜缘区域有40.8 +/- 12.0（平均值 +/- 标准差）个细胞结合4G10.3。在进行3 mm的清创伤口后，结合4G10.3的细胞数量在损伤后两天增加到77.0 +/- 16.9，并在三天后恢复到对照水平。热烧伤后，每角膜缘区域结合4G10.3的细胞数量在损伤后18小时、1天、2天、3天和7天分别为36.2 +/- 11.2、68.8 +/- 15.8、85.4 +/- 15.4、104.6 +/- 13.8和88.0 +/- 40.2。50 kD蛋白和4G10.3抗体提供了角膜缘基底细胞的生化和免疫标记，推测这些细胞为角膜上皮干细胞。
