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目的 评价深低温冷冻保存兔眼角膜缘干细胞活性。方法 12只 (12眼 )新西兰白兔 ,制备 2mm周边角膜和 2mm球结膜的环形浅层角膜缘植片及角膜干细胞缺乏眼表疾病模型。角膜缘植片通过程序降温仪程序降温后 - 196℃深低温保存 ,30天和 6 0天后行角膜缘自体移植。组化及免疫组化证实深低温保存的角膜缘干细胞活性。结果 角膜干细胞缺乏导致角膜上皮愈合延迟 ,上皮结膜化 ,PAS染色阳性 ,AE5染色弱阳性 ;基质水肿 ,角膜血管化。深低温保存角膜缘干细胞自体移植能促进角膜上皮愈合 ,为角膜表型 ,PAS染色阴性 ,AE5染色阳性。基质水肿逐渐吸收 ,新生血管消退、变细。结论 深低温保存法能保存角膜缘干细胞活性 ;能随时按需为临床提供活性角膜缘材料 ;为深低温保存角膜缘干细胞移植治疗临床眼表疾病提供了实验依据。
Objective To evaluate the cryogenic cryopreservation of rabbit limbal stem cells. Methods Twelve eyes (12 eyes) of New Zealand white rabbits were used to prepare the ring-shaped shallow limbal limbal graft with 2mm corneal conjunctiva and 2mm corneal conjunctiva. The corneal stem cells lacked the ocular surface disease model. The corneal limbal grafts were cryopreserved by a program of cryopreservation. After cryopreservation at 196 ℃, corneal limbal autografts were performed 30 days and 60 days later. Histological and immunohistochemistry confirmed the survival of limbal stem cells after cryopreservation. Results The lack of corneal stem cells resulted in delayed healing of corneal epithelium, epithelial conjunctivalization, positive PAS staining, weak positive AE5 staining, stromal edema and corneal vascularization. Cryopreservation limbal stem cells autograft can promote corneal epithelial healing, corneal phenotype, PAS staining negative AE5 staining positive. Gradually absorbed matrix edema, neovascularization subsidence, thinning. Conclusion Cryopreservation method can preserve the limbal stem cell activity. It can provide active limbal material as needed. It provides the experimental evidence for cryopreserved limbal stem cell transplantation in the treatment of clinical ocular surface disease.