角膜缘干细胞移植后供体干细胞的检测与分析

Detection and Analysis of Donor Stem Cells after Limbal Stem Cell Transplantation

目的观察新西兰兔角膜缘干细胞移植后供体干细胞数量的变化与角膜上皮化的关系,为角膜缘干细胞移植手术效果提供理论依据。方法新西兰兔30只,分为自体移植和异体移植两组,建立角膜缘干细胞缺损动物模型,分别进行自体和异体角膜缘干细胞移植。术后1周、1月、3月空气栓塞各处死5只,取包括角膜缘的角膜标本,免疫组织化学p63蛋白抗体检测供体干细胞的存活数量和增殖分化状况,比较两组的移植效果。结果自体移植术后1周,供体干细胞仍然局限于受体角膜缘;1个月后,上皮细胞覆盖角膜全层,但是层次非常少;3个月后,干细胞的储备数量基本上恢复到正常水平,角膜完全上皮化。异体移植1周,干细胞的数量有一定的减少;1个月后,角膜缘干细胞储备数量明显减少;3个月后,角膜缘已经检测不到干细胞。结论干细胞的储备数量和增殖分化状况直接影响到角膜上皮化的结局;有效的干细胞检测结果可以为临床角膜缘干细胞移植效果提供依据,p63蛋白抗体可以作为一个检测角膜缘干细胞的特异指标。

Objective To investigate the correlation between the survival of donor-derived limbal stem ceils and the reepithelization of cornea, and to provide evidence and evaluation for limbal stem cell transplantation. Methods Thirty New-Zealand rabbits were divided into two groups： me autograft group. Corneal epithelial stem cells were obtained by autograft and allograft limbal stem cell transplantation. The corneal samples were analyzed by immunohistochemistry（SP method ）. Results In autograft group, the donor stem ceils were still confined to the limbus during the first week; whereas a well-differentiated corneal epithelium was recognized in the first month with ceils proliferatition, differention and migration to the central part of the cornea but formed only two to three layers; 3 months later, a normal stratified corneal epithelium with five to six cell layers was found. In allograft group, the marked stem cells decreased during the first week, and nearly no epithelium covered in the first month. In the third month, the cornea was accompanied with chronic inflammation, persistent defects, stromal scaring, neovascularization, and stem ceil disappeared. Conclusion The amount of limbal stem cells has a direct influence uport the outcome of reepithelization of cornea. Effective detection of survival of limbal stem cells provides an essential evidence for the result of limbal stem cell transplantation, p63 is a specific marker for the signal and identified of limbal stem cells.