方法：选择新鲜猪角膜，以13mm直径环钻制取角膜标本，依次放入四种冷冻保护液中冷平衡，“简化四步法”程序降温后放入液氮中保存，使用前40℃水浴复温。地塞米松作用组以同法制备植片，前三步冷平衡同前，最后分别放入含0.01，0.03，0.05mg/mL地塞米松保护液中4℃孵育18h，再程序降温液氮冻存。BALB/c小鼠50只，随机分为阳性对照组(新鲜组)、程序冻存组、地塞米松程序冻存组Ⅰ、地塞米松程序冻存组Ⅱ和地塞米松程序冻存组Ⅲ，每组10只。各组角膜植入到BALB/c小鼠背部皮下，术后14d取出，做石蜡包埋，HE染色、CD25和FasL免疫组化染色，光镜观察。

结果：术后14d剖取角膜植片，见植片与周围组织粘连，植片明显肿胀，半透明，色微黄。HE染色结果：新鲜组角膜植片全层有大量淋巴细胞浸润； 程序冻存组角膜浸润细胞较新鲜组减少，多位于内皮和上皮层分布； 地塞米松作用各组细胞浸润数最少。免疫组化显示：新鲜组植片CD25⁺和FasL⁺浸润细胞数最多，与程序冻存组、各地塞米松作用组比较有明显统计学差异。程序冻存组CD25⁺和FasL⁺浸润细胞数多于各地塞米松作用组，比较有显著性差异。各地塞米松作用组CD25⁺和FasL⁺浸润细胞数差异比较无统计学意义。

结论：地塞米松作用组角膜免疫原性最低。各地塞米松作用组角膜免疫原性在0.01～0.05mg/mL范围内无浓度依赖效应。

METHODS: Fresh porcine corneas were drilled down by 13mm-diameter trephine. Then the samples were put into four kinds of cryoprotectants in turn for cooling equilibrium with “simplified four-step” method and then stored in liquid nitrogen. All samples were thawed in water at 40℃ before being used. The samples treated with dexamethasone were made and the preceding three cooling steps were same. But the samples were additionally incubated in 0.01, 0.03, 0.05mg/mL dexamethasone for 18h at 4℃ before the last cooling step. Totally 50 BALB/c mice were randomly divided into 5 groups including 10 mice in each group: the control group; routine cooling group which were treated through four cooling steps; dexamethasone group I, dexamethasone group II and dexamethasone group III which were treated with 0.01, 0.03, 0.05mg/mL dexamethasone successively. After treatment, corneal samples were transplanted into the hypodermis on the back of mice. After 14d, the samples were taken out, imbedded in paraffin, and stained by HE and CD25/FasL immunohistochemistry. All the samples were investigated under light microscope.

RESULTS: On the 14th day, all the samples were taken out. We found the samples which adhered to surrouding tissues swelled obviously and showed translucent and a bit yellow. Through HE staining, fresh corneal samples in the control group were infiltrated with large amount of lymphocytes throughout each layer; the amount of infiltrating cells in the routine cooling group was less than the control group in which most cells were distributed in endodermis and epitheliums. In the dexamethasone-treated groups, the amount of infiltrated cells were the least. Immunohistochemical results showed that compared with the the routine cooling group and dexamethasone-treated groups, the amount of CD25 and FasL in the control group was the most, the difference was significant. Compared with dexamethasone-treated groups, the amount of CD25 and FasL positive infiltrating cells in the routine cooling group were more. ALL the differences were significant. But there was no significant difference in the amount of CD25 and FasL positive infiltrating cells among each dexamethasone treated group.

CONCLUSION: Corneas in dexamethasone-treated groups showed the lowest immunogenicity. Corneal immunogenicity was irrelevant with the concentration of dexamethasone at the range of 0.01-0.05mg/mL.