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目的 通过光/电镜酶组织细胞化学方法测定兔角膜内皮细胞酶活性,探讨真空冷冻干燥保存法(冻干法)对于兔角膜内皮细胞活性的影响。方法 按随机及配对原则将兔眼分为新鲜对照组、冷冻对照组、冻干实验组。对冷冻组进行角膜深低温冷冻保存。对冻干组进行真空冷冻干燥保存。将冷冻及冻干保存的兔角膜片分别复水及复温后,与新鲜的角膜片同时进行三磷酸腺苷酶(AT Pase)、琥珀酸脱氢酶(SDH)的光镜及电镜酶组织细胞化学染色,观察三组酶的活性是否有差异。结果 在新鲜组、冷冻组、冻干组中,ATPase及DSH的酶组织化学染色在光镜观察下均呈阳性反应。三组样本内皮细胞ATPase和SDH酶的平均积分光密度值(IOD)差异具有统计学意义(P <0 .0 1)冻干组活性较低。电镜酶组织细胞化学染色见三组样本角膜内皮细胞膜上均有电子致密颗粒沉着。结论 真空冷冻干燥保存法可使离体角膜内皮细胞保持一定的活性。与新鲜及冷冻保存的兔角膜相比,活性较低。但可以推测冻干法有望成为一种新的角膜长期保存方法
Objective To determine the enzyme activity of corneal endothelial cells in rabbit by light / electron microscopy and to investigate the effect of freeze-drying method on the activity of rabbit corneal endothelial cells. Methods Rabbit eyes were divided into fresh control group, frozen control group and freeze-dried experimental group according to randomization and pairing principle. Frozen corneal cryogenic cryopreservation. The freeze-dried group was vacuum freeze-dried preservation. Frozen and lyophilized rabbit corneas were rehydrated and rewarmed, respectively, and fresh corneal slices were simultaneously treated with AT Pase, succinate dehydrogenase (SDH) light microscopy and electron microscopy of histochemical staining , Observe the activity of the three groups whether there is a difference. Results The enzymatic histochemical staining of ATPase and DSH in fresh group, frozen group and lyophilized group all showed positive reaction under light microscope. The mean integral optical density (IOD) values of three groups of samples of endothelial cells ATPase and SDH enzyme were statistically significant (P <0. 01) lyophilized group activity is low. Electron microscopy showed that there were electron-dense particles on the corneal endothelial cell membrane in three groups of samples. Conclusion The method of vacuum freeze-drying preserves the corneal endothelial cells in vitro. Compared with fresh and cryopreserved rabbit cornea, the activity is lower. But it can be speculated that lyophilization is expected to become a new method of long-term corneal preservation