深低温保存角膜缘干细胞羊膜片的异体移植

Experimental hetero-grafting of cryopreserved corneal limbus epithelium on the upper parts of the amnion

目的 :探讨深低温冷冻保存角膜缘干细胞羊膜片的活性及异体移植疗效。方法 :将兔角膜缘干细胞移植到羊膜上 ,经简化程序降温后置 - 196℃深低温保存 ,并将兔对侧眼制成碱烧伤模型。第 30天～第 4 5天后抽样取出冷冻保存的植片分别行电镜检查及兔角膜缘干细胞异体移植 ,并收稿日期 :2 0 0 2 -0 3 -18;修回日期 :2 0 0 2 -0 7-15基金项目 :浙江省教委课题基金 (sjy0 0 0 0 6)。作者简介 :韩真真 ( 195 0 -) ,女 ,浙江温州人 ,主任医师。研究方向 :眼表疾病和角膜病。通信作者 :韩真真 (E -mail:zuixianmei@sina .com)。于术后第 1个月时进行印迹细胞学检查。结果 :电镜检查显示冷冻保存的角膜缘干细胞超微结构形态正常 ,线粒体轻微水肿。移植术后第 1个月时 ,角膜上皮稳固愈合 ,混浊角膜渐转透明 ,新生血管减少 ,角膜印迹细胞学检查显示角膜上皮细胞正常 ,未见杯状细胞。结论 :深低温保存的角膜缘干细胞羊膜片能有效用于眼表的重建 。

Objective:To investigate the activation of cryopreserved corneal limbus epithelium on the upper parts of amnion and the effect of hetero grafting.Methods:Three 120 ° grafts from one eye of each rabbit,which included 1.5 mm of corneal limbus epithelium and 2 mm of conjunctiva,were transplanted onto the upper parts of 2 cm×1 cm of the amnion by 10 0 nylon suture. The graft was persevered in a 5% admixture for 20 min and then in a 7.5% admixture for 20 min with white protein as menstruum and DMSO as coolant. The tissue was then placed in refrigeration in subsequent steps at -4 ℃ for 10 min,-12 ℃ for 10 min,-80 ℃ for 10 min and finally cryopreserved at -196 ℃. The other eyes that were examined had ocular surface disorders derived from alkali chemical burns:11 mm round filter paper was dipped in 10% NaOH for 5 sec,then placed on the ocular surface of the other eye for 40 sec,then cleaned in 0.9% NaCl for 60 sec. After 30 days and 45 days,the two grafts were hetero transplanted onto the damaged ocular surfaces while the other graft was evaluated under electron microscopy. After surgery,corneal transparency,corneal graft rejection and neovascularization were examined. Corneal impression cytology was performed after 1 min.Results:Electron microscopic examination revealed that the cells of simple cryopreserved corneas were intact except for swollen mitochondria. Follow up at 1 min revealed that the epithelium had survived,corneal clouding had become transparent,and neovascularization had decreased. Corneal impression cytology showed that the corneal epithelium was normal.Conclusion:The cryopreserved corneal limbus epithelium on the upper parts of the amnion is suitable for a reconstructed ocular surface but activation of the graft using simpler procedures should be re evaluated.