兔角膜器官培养保存与应用的研究

Experimental study on corneal organ culture preservation and its application

目的探讨建立角膜器官培养保存的方法、保存后角膜的特性及其用于移植的可行性。方法选用特定的胎牛血清保存液,密闭保存36只成年新西兰兔角膜于32℃恒温箱中;在保存后1-4周的不同时间点,将角膜从保存液取出转入高渗透压的脱水液中脱水48h 备用;而后对比保存前后角膜内皮细胞密度、角膜厚度变化,并观测角膜内皮组织学特性和保存中污染情况;器官培养保存后的兔角膜做同种异体穿透性角膜移植,观测术后1周内植片情况;使用荧光标记的抗 MHC-Ⅱ分子抗体标示保存后的角膜中树突状细胞,观测其分布和保存后变化特点。结果成功建立了角膜器官培养保存方法,保存中角膜的污染率约8.3%;保存4周内的兔角膜脱水后均透明;保存第3周时,角膜后弹力层周边区有细小皱褶;保存第4周时,全角膜有明显皱褶;器官培养保存的兔角膜内皮细胞密度随保存时间延长而下降,保存4周后兔角膜内皮细胞密度平均下降15.7%;兔角膜厚度随保存时间延长而增加,保存第4周时,角膜中央厚度可达0.7mm;组织学观察可见角膜内皮细胞与后弹力层贴附紧密;树突状细胞分布于角巩膜缘上皮层,保存至第3周时全部消失;同种异体角膜移植术后7d 内植片透明,无原发失败。结论本研究建立的角膜器官培养保存方法可使保存的角膜在一定时间内维持活性,角膜的免疫原性下降。

Objective To develop a optimal method of corneal organ culture preservation, to evaluate the characteristics of organ cultured cornea and to examine the feasibility of application of organ cultured corneas. Methods Thirty-six adult New Zealand rabbit corneas were stored in the closed storage system consisting of fetal calf serum and were transferred to a hyperosmosis dextran-containing medium for 48 hours＇ deswelling for further experiment after storage for 1, 2, 3 and 4 weeks. The measurement of corneal endothelium density and central thickness was performed prior to and after the storage. The morphology and the contamination of preserved cornea, as well as allogenic grafts＇ conditions within 7 days after corneal transplantation were also observed. The class Ⅱ MHC antigen immunofluorescence marker was performed for the examination of corneal dendritic cells by confocal microscopy. Results The corneal organ culture preservation was successfully established. The contamination rate was approximately 8.3%. All of the rabbit corneas stored within 4 weeks remained transparent. There were little folds in peripheral descemet＇s membrane when the storage time was up to 3 weeks and the whole descemet＇s membrane folds was seen when the corneas had been preserved for 4 weeks. The density of corneal endothelium decreased when the preservation time was extended. The loss of corneal endothelium preserved up to 4 weeks was 15.7%. Correlation existed between the thickness of center cornea and the preservation time. The average central thickness of the cornea was 0.7 mm after 4 weeks. The light micrographs showed that the endothelium layer still covered the descemet＇s membrane completely after 4 weeks. The dendritic cells resided in the limbus epithelium and they were lost after stored for 3 weeks. After allogenic corneal transplantation, the grafts retained transparence within 7 days postoperatively, which indicated that there was no primary failure of grafts. Conclusion Corneal organ culture preservation can ensure the normal function of cultured cornea within a certain preservation time and offer some advantages, e.g. the decrease of corneal immunogenicity.