Li Wei, Hayashida Yasutaka, Chen Ying-Ting, He Hua, Tseng David Y, Alonso Morgan, Chen Szu-Yu, Xi Xinghua, Tseng Scheffer C G
TissueTech, Inc., and the Ocular Surface Center, 7000 SW 9th Avenue, Miami, FL 33173, USA.
Invest Ophthalmol Vis Sci. 2008 Jan;49(1):154-62. doi: 10.1167/iovs.07-0883.
Squamous metaplasia is a pathologic process that frequently occurs in nonkeratinized stratified ocular surface epithelia. The mechanism for this occurrence is largely unknown except for vitamin A deficiency.
Human limbal explants were cultured under airlift with or without p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 or in a submerged manner for different durations up to 2 weeks. Epithelial cell proliferation, differentiation, limbal stem cell maintenance, and expansion were studied using certain markers such as Ki67, p63, K10 and K12 keratins, filaggrin, Pax6, ABCG-2, and Musashi-1. Expression of phospho-p38 MAPK and its downstream transcription factors, C/EBPalpha and C/EBPbeta, were studied by immunohistochemistry. Epithelial cells harvested from explants after 2 weeks of culturing under different conditions were seeded onto 3T3 feeder layers and cultured for 12 days. The differentiation of clonal epithelial cells was investigated by double staining to K12 and K10 keratins.
The squamous metaplasia model was successfully created by culturing human limbal explants at an air-liquid interface (airlift) for 2 weeks. Increased stratification and hyperproliferation only happened in the limbal, but not the corneal, epithelium in airlift, but not submerged, cultures. Epithelial proliferation was associated with a transient increase of limbal epithelial stem cells. Abnormal epidermal differentiation-evidenced by positive expression of K10 keratin in suprabasal cells and filaggrin in superficial cells-ensued. Clones generated from epithelial cells harvested from airlift culture only expressed K12 keratin without K10. As early as 2 days in airlift cultures, p38 expression emerged in limbal basal epithelial cells and gradually extended to the cytoplasm and nuclei. Furthermore, addition of the p38 inhibitor SB203580 abolished abnormal epidermal differentiation without affecting limbal epithelial proliferation. Expression of C/EBPalpha and C/EBPbeta, downstream of the p38 MAPK signaling pathway, was strongly induced by airlift culture and partially was inhibited by SB203580.
Dryness resulting from exposure activates p38 MAPK signaling coupled with abnormal epidermal differentiation without intrinsic alteration of stem cells in the limbus. On the ocular surface, p38 inhibitors may have the potential to revert the pathologic process of squamous metaplasia induced by dryness.
鳞状化生是一种常见于非角化复层眼表上皮的病理过程。除维生素A缺乏外，其发生机制 largely unknown。
人角膜缘外植体在气升培养条件下，添加或不添加p38丝裂原活化蛋白激酶（MAPK）抑制剂SB203580，或以浸没方式培养不同时长，最长2周。使用某些标志物，如Ki67、p63、K10和K12角蛋白、丝聚蛋白、Pax6、ABCG - 2和Musashi - 1，研究上皮细胞增殖、分化、角膜缘干细胞维持和扩增情况。通过免疫组织化学研究磷酸化p38 MAPK及其下游转录因子C/EBPα和C/EBPβ的表达。将在不同条件下培养2周后的外植体收获的上皮细胞接种到3T3饲养层上，培养12天。通过对K12和K10角蛋白进行双重染色，研究克隆上皮细胞的分化情况。
通过在气液界面（气升）培养人角膜缘外植体2周，成功建立了鳞状化生模型。分层增加和过度增殖仅发生在气升培养而非浸没培养的角膜缘上皮，而非角膜上皮。上皮增殖与角膜缘上皮干细胞的短暂增加有关。出现异常的表皮分化，表现为基底上层细胞中K10角蛋白和表层细胞中丝聚蛋白阳性表达。从气升培养收获的上皮细胞产生的克隆仅表达K12角蛋白，而不表达K10。早在气升培养2天时，p38表达出现在角膜缘基底上皮细胞中，并逐渐扩展到细胞质和细胞核。此外，添加p38抑制剂SB203580可消除异常的表皮分化，而不影响角膜缘上皮增殖。p38 MAPK信号通路下游的C/EBPα和C/EBPβ的表达在气升培养时被强烈诱导，部分被SB203580抑制。
暴露导致的干燥激活p38 MAPK信号通路，伴有异常的表皮分化，而角膜缘干细胞无内在改变。在眼表，p38抑制剂可能具有逆转干燥诱导的鳞状化生病理过程的潜力。
