不同降温速度对保存的角膜内皮细胞活性的影响  

Survival of cornea endothelial cell via long-term cryopreservation

作　　者：贾艳红[1] 王丽娅[1] 杜连心[1] 赵承彦[1] 

机构地区：[1]河南省眼科研究所

出　　处：《眼科研究》2007年第5期343-346,共4页Chinese Ophthalmic Research

基　　金：河南省省属科研机构研究开发专项资金资助(0641130310)

摘　　要：目的通过调节程序控制降温仪的降温速度保存兔角膜,以提高保存角膜的内皮细胞的存活率(ESR)。方法供体角膜150只随机分为对照组和实验组。对照组不做冷冻处理;实验组冷冻147只角膜,带巩膜缘1~2mm的角膜片分别在4种不同浓度梯度的冻存液中预处理各10min,再经过两个阶段(0^-18℃,-18^-80℃)49种不同程序逐步降温至-80℃,最后放入液氮中保存。1个月后水浴复苏供体角膜,洗脱冻存液、染色、固定,行镜下形态学观察并计算角膜内皮细胞存活率。结果镜下观察第一阶段降温速度为2℃/min、第二阶段降温速度为5℃/min时的方法较好,计数细胞的存活率达到75%以上,与其他不同速度下比较差异有统计学意义(P<0.05)。结论不同阶段不同降温速度下,深低温保存兔角膜对角膜的内皮细胞存活率有明显影响。Objective To investigate the survival rate of cornea endothelial cells preserved by different procedure of cryopreservation. Methods 150 rabbit corneas were divided into normal control group and experimental group,and 147 corneas in experimental group were subdivided into 49 groups according to different frozen procedure. Four vials of preservative solution were prepared in different concentrations. The corneal buttons with a l - 2 mm sclera rim were soaked in above four preservative solutions for 10 rain respectively and then were transferred into programmed freezer. The corneal buttons were frozen in different controllablerated freezer to -80 ℃ ,and were stored in liquid nitrogen. One month later,the corneal buttons were thawed in water bath, and preservative solution was removed from the vial of cornea. The corneal buttons were eluted in DMSO containing 25% FBS solution. Corneal endothelium was dual stained using a 0.25% solution of Trypane blue and 0.4% stock solution of Alizarin red S（ ph4. 2）. 2..5% solution glutaraldehyde was used to fix each corneal buttons for morphology observation. Results When the lowingtemperature rate was 2.0 ℃/min in the first phase and 5.0 ℃/min in the second phase,adumbration of endothelial cell presented red staining,and intercellular tight junction was identified. No blue nucleus were seen. Corneal endothelium cells showed over 75% survival rate. But in other procedure, adumbration of endothelial cell showed red color and round in shape, and many blue-stained nucleus,stripping of endothelial cell and naked nucleus were found. A significant difference was found in survival rate of cryopreservated-corneal endothelial cells under the condition of 2.0 ℃/min in the first phase and 5.0 ℃/min in the second phase in comparison with other different lowing-temperature velocity（P 〈 0. 05 ）. Conclusion The lowing-temperature velocity and phases during the long-term cryopreservation affect the survival of cornea endothelial cell.

关 键 词：角膜内皮细胞 降温程序 深低温保存 

分 类 号：R779.65[医药卫生—眼科]