Cell cycle protein expression and proliferative status in human corneal cells.
Cell cycle protein expression and proliferative status in human corneal cells.
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发表时间：
1996-03
影响因子：
4.4
通讯作者：
N. Joyce;B. Meklir;Steven J. Joyce;J. Zieske
中科院分区：
文献类型：
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作者：
N. Joyce;B. Meklir;Steven J. Joyce;J. Zieske
目的研究细胞周期相关蛋白在人角膜和角膜缘上皮及角膜内皮原位组织中的相对表达，将细胞周期相关蛋白的染色模式与已知角膜和角膜缘上皮细胞的增殖状态相关联，并通过与角膜和角膜缘上皮细胞的染色模式进行比较，确定角膜内皮原位细胞的相对增殖状态。方法将6周及17、27、37、53、66、67岁供者的角膜保存在Optisol中，在死亡后24 ~ 36小时内冰敷，并立即新鲜冷冻。使用特异性识别以下细胞周期相关蛋白的商业抗体制备横向6微米角膜切片进行间接免疫荧光定位：细胞周期蛋白D、E、A和B1；蛋白激酶p33cdk2和p34cdc2；以及活跃循环细胞的标记物Ki67。结果角膜、角膜缘上皮细胞和角膜内皮蛋白激酶p33cdk2、p34cdc2和细胞周期蛋白B1均呈阳性。细胞周期蛋白D、E、A和Ki67的染色模式不同，取决于细胞的相对增殖状态。终末分化的非循环角膜上皮亚基底细胞没有明显的细胞周期蛋白D、E、A或Ki67染色。一些角膜上皮基底细胞呈核染色，特别是cyclin D和Ki67，表明在这一再生细胞层中存在活跃的循环细胞。在角膜周围上皮中，细胞周期蛋白D、E和A细胞质染色呈阳性的基底细胞的相对数量随着靠近角膜缘而增加。在这个区域内，偶见Ki67阳性细胞核。含有慢循环干细胞群的边缘基底细胞细胞质中细胞周期蛋白D、E和a呈阳性染色。少量基底细胞可见细胞周期蛋白D和Ki67的核染色。边缘上基底层细胞偶有Ki67阳性染色。角膜内皮，被认为是一个不更新的群体，表现出与角膜缘基底细胞相似的染色模式，除了没有在标本中观察到Ki67染色。结论：所有角膜、角膜缘上皮细胞和角膜内皮细胞均表达蛋白激酶p33cdk2、p34cdc2和细胞周期蛋白B1。细胞周期依赖蛋白、细胞周期蛋白D、E和A以及Ki67的相对染色模式作为区分已退出细胞周期的终末分化上皮基底上细胞、主动循环上皮基底细胞和缓慢循环的缘基（干）细胞的标记物。角膜内皮的染色模式与角膜缘基底细胞的染色模式最接近，表明内皮细胞停留在g1期，未退出细胞周期。
PURPOSE To determine the relative expression of cell cycle-association proteins in human corneal and limbal epithelium and corneal endothelium in situ, to correlate staining patterns of cell cycle-associated proteins with known proliferative status of corneal and limbal epithelial cells, and to determine the relative proliferative status of corneal endothelial cells in situ by comparing their staining patterns with those of corneal and limbal epithelial cells. METHODS Corneas from donors 6 weeks and 17, 27, 37, 53, 66, and 67 years of age were preserved in Optisol, received on ice within 24 to 36 hours of death, and immediately fresh frozen. Transverse 6-micron corneal sections were prepared for indirect immunofluorescence localization using commercial antibodies that specifically recognize the following cell cycle-associated proteins: cyclins D, E, A, and B1; protein kinases p33cdk2 and p34cdc2; and Ki67, a marker of actively cycling cells. RESULTS All cells of the corneal and limbal epithelium and corneal endothelium stained positively for protein kinases, p33cdk2 and p34cdc2, and for cyclin B1. Staining patterns for cyclins D, E, and A and for Ki67 differed depending on the relative proliferative status of the cells. Terminally differentiated, noncycling corneal epithelial subrabasal cells did not stain significantly for cyclins D, E, or A, or for Ki67. Some corneal epithelial basal cells showed nuclear staining, particularly for cyclin D and Ki67, indicating the presence of actively cycling cells in this regenerative cell layer. In peripheral corneal epithelium, the relative number of basal cells with positive cytoplasmic staining for cyclins D, E, and A increased with proximity to the limbus. Within this region, an occasional nucleus stained positively for Ki67. Limbal basal cells, which contain a slow-cycling stem cell population, stained positively for cyclins D, E, and A within the cytoplasm. Nuclear staining for cyclin D and Ki67 was observed in a few basal cells. Occasional cells within the suprabasal layers of the limbus stained positively for Ki67. The corneal endothelium, considered a nonrenewing population, exhibited staining patterns similar to those of limbal basal cells, except that in no specimen was Ki67 staining observed. CONCLUSIONS All corneal and limbal epithelial and corneal endothelial cells express protein kinases, p33cdk2 and p34cdc2, and cyclin B1. Relative staining patterns of the cell cycle-dependent proteins, cyclins D, E, and A, and of Ki67 acted as markers to distinguish terminally differentiated epithelial suprabasal cells that have exited the cell cycle, actively cycling epithelial basal cells, and slowly-cycling limbal basal (stem) cells. Staining patterns of the corneal endothelium most closely corresponded to those of limbal basal cells, suggesting that endothelial cells are arrested in G1-phase and have not exited the cell cycle.