Joseph A, Powell-Richards A O R, Shanmuganathan V A, Dua H S
Larry A Donoso Laboratory for Eye Research, Division of Ophthalmology and Visual Sciences, University of Nottingham, Nottingham NG7 2UH, UK.
Br J Ophthalmol. 2004 Mar;88(3):393-8. doi: 10.1136/bjo.2003.018481.
To determine the immunohistochemical characteristics of putative corneal epithelial stem cells remaining on limbal explants maintained in culture.
Human limbal explant cultures were generated from 25 residual corneoscleral donor rims following penetrating keratoplasty. Serial sections of these explants were studied using immunohistochemical techniques with a panel of antibodies, on day 0 and 1, 2, and 3 weeks.
The number of epithelial cells expressing cytokeratin 19 and vimentin increased with duration in culture, while the number of cells expressing cytokeratin 3 decreased. Connexin 43 expression was lost by 1 week in culture. p63 was expressed by cells that had migrated around the explant and the number of p63 positive cells decreased with longer duration in culture. The explants were initially negative for Ki67, but the epithelial cells were positive at 1 week, and expression of Ki67 was progressively lost with increasing duration in culture. The initial uniform staining of the epithelium for epidermal growth factor receptor and alpha enolase remained unchanged at 3 weeks.
There is an expansion of less differentiated (cytokeratin 3 negative and CK19/vimentin positive) epithelial cells on corneoscleral explants maintained in culture for 3 weeks. The pattern of expression of p63 noted in this study does not support the suggestion that it is a marker of limbal stem cells. The decline in p63 and Ki67 expression among the epithelial cells of the cultured explant button implies that as the epithelial sheet outgrowing from the explant button reaches confluence, the proliferative status of the cells remaining on the explant button declines. These findings are of clinical relevance as explants of limbal tissue are used in limbal stem cell transplantation. There is no information available to date on the fate of epithelial cells on such explants. This study provides some insight into this and suggests that an expansion of the stem cell pool or its progeny may occur in limbal explants.
确定培养的角膜缘外植体上残留的假定角膜上皮干细胞的免疫组织化学特征。
穿透性角膜移植术后，从25个剩余的角膜巩膜供体边缘获取人角膜缘外植体培养物。在第0天、第1周、第2周和第3周，使用一组抗体通过免疫组织化学技术对这些外植体的连续切片进行研究。
随着培养时间的延长，表达细胞角蛋白19和波形蛋白的上皮细胞数量增加，而表达细胞角蛋白3的细胞数量减少。培养1周后连接蛋白43表达消失。p63由在外植体周围迁移的细胞表达，且随着培养时间延长，p63阳性细胞数量减少。外植体最初Ki67呈阴性，但上皮细胞在1周时呈阳性，且随着培养时间延长，Ki67表达逐渐消失。表皮生长因子受体和α烯醇化酶的上皮初始均匀染色在3周时保持不变。
在培养3周的角膜巩膜外植体上，低分化（细胞角蛋白3阴性和CK19/波形蛋白阳性）上皮细胞有所扩增。本研究中观察到的p63表达模式不支持其作为角膜缘干细胞标志物的观点。培养的外植体纽扣上皮细胞中p63和Ki67表达的下降意味着，随着从外植体纽扣长出的上皮片达到汇合，留在外植体纽扣上的细胞的增殖状态下降。这些发现具有临床相关性，因为角膜缘组织外植体用于角膜缘干细胞移植。迄今为止，尚无关于此类外植体上上皮细胞命运的信息。本研究对此提供了一些见解，并表明角膜缘外植体中可能发生干细胞池或其后代的扩增。
