深低温冷冻保存角膜的实验研究

Experimental study of quick freezing at extremely low temperature on preservation of corneas

关键词:  角膜  低温保存  显微镜

Key words:  corneae  cryopreservation  microscopy

摘要:

验证二甲基亚砜(DMSO)是否是目前最好的角膜冷冻保护剂，探讨乙醇可否替代DMSA。［方法］16只新鲜兔角膜随机分为DMSO组和乙醇组。将角膜于4 ℃分别浸入浓度递增的两组保护液中，冷平衡后，按1.5～2 ℃/min的速度降温至-30 ℃后，再以5 ℃/min的速度降温至-80 ℃，然后,将标本瓶投入液氮中冷冻保存角膜5 d。取出后置于50 ℃流动的热水中快速复温，脱保护剂。每组各取6只角膜，经锥兰-茜素红染色后，用光学显微镜计数并计算每只角膜平均内皮细胞存活率(ESR)。另2只角膜，用2.5%戊二醛溶液固定，分别用扫描电镜和透射电镜观察内皮细胞超微结构。［结果］DMSO组和乙醇组平均ESR分别为(96.18±2.49)%和(92.55±6.24)%（P<0.05）。扫描电镜下，DMSO组角膜内皮细胞呈六角形，细胞完整似新鲜角膜，细胞间隙稍宽。乙醇组角膜内皮亦呈六角形，但细胞间隙明显增宽。透射电镜显示两组角膜内皮细胞均有线粒体肿胀，内质网扩张及胞质内空泡形成，但乙醇组改变更明显。［结论］本研究提示乙醇对角膜冷冻保存有一定效果，但尚不如DMSO

Abstract:

To verify whether dimethyl sulfoxide(DMSO)was the best cryopretective agent and to study whether ethanol could replace DMSO. ［Method］Sixteen fresh corneas were divided into DMSO group and ethanol group.Corneas were submersed in the above two kinds of solution at 4 ℃.The equilibrium time of each solution was ten minutes.Then adjusted the level between the vials and liquid nitrogen in order to get the cooling rate of 1.5～2.0 ℃/min. When the temperature was -30 ℃,cooling rate began to change to 5 ℃/min until -80 ℃ was reached. Finally the vials were preserved in liquid nitrogen`for 5 days.The vials were thawed at 50 ℃ running water within 1 minute.Then six corneas of each group were dyed by 0.25% typan blue and were dyed by 0.2% alizarin red S.Endothelial cells were examined by the light microscopy and the mean ESR' of each cornea was calculated. The left corneas of each group were put into the 2.5% glutaraldehyde solution and evaluated by the scanning and transmission electron microscopy to observe their ultrastructure. ［Result］The mean ESR in DMSO group was (96.18±2.49)%.It was (92.55±6.24)% in ethanol group（P<0.05）.Under the scanning electron microscope, endothelial cells of two group were hexagonal.Intracelluar spaces were much wider in ethanol group.Under the transmission electron microscope,mitrochondrial swelling, endoreticulum dilatation and cystic formation appeared in the endothelium of both groups, but these changes in ethanol group were more prominent