透明质酸钠在甘油冷冻保存角膜中的应用

The use of sodium hyaluronate in cryopreservation of cornea

目的评价甘油冷冻保存角膜的效果以及透明质酸钠对保存角膜内皮细胞(CEC)的保护作用。方法20只兔眼角膜片随机分为两组。对照组角膜直接放入纯甘油中,-25℃保存,实验组则在内皮面均匀黏附透明质酸钠后再放入甘油中低温保存。保存2个月后两组均接受CEC锥蓝-茜素红活性染色检查、电镜检查以及穿透性角膜移植(PKP)实验,以检验保存效果。结果两组CEC均能保持细胞活性,实验组的保存效果要好于对照组,其CEC密度为3067·9±127·6/mm2,死亡率为4·2%±3·4%,且行PKP较对照组有更高的成功率和CEC密度。结论甘油冷冻能够保持CEC的活性,透明质酸钠能进一步增进保存效果,改良后的甘油冷冻保存方法有希望成为一种方便、经济、有效的角膜保存方法。

Objective To assess the effect of cryopreservation of cornea in glycerol, to analyze the protective effect of sodium hyalu ronate on corneal endothelial cells （CEC） during preservation, and to find out a convenient and cheap method for the storage of comea. Methods 20 rabbit corneas were divided into two groups randomly. The corneas of control group were stored directly in pure glycerol at -25℃, while in the experimental group sodium hyaluronate was evenly anointed on the corneal endothelial surface of the cornea before be ing cryopreserved in glycerol. 2 months later, the effect of preservation for both groups was examined with trypan blue combined with alizarin red staining （viability staining） for corneal endothelial cells, electron-microscope examination for preserved corneas, and rabbit experimental penetrating keratoplasty （PKP） with the long-term cryopreserved corneas from both the experimental and control groups. Results CEC viability could be preserved by both methods. CEC trypan blue combined with alizarin red staining （viability staining） showed that the CEC density of cornea in the experimental group was 3 067. 9±127. 6/mm^2, which was higher than that of control group （2 899. 9± 134. 0/mm^2）. The mortality of CEC in experimental group was 4.2%±3.4%, lower than that in control group （13. 8%±2. 2%）. Suceessful rate of PKP was higher in the experimental group with higher CEC density. Conclusion Cryopreservation with glycerol can preserve the viability of CEC. Sodium hyaluronate can reduce the damage to endothelium in the preserving procedure and can obviously im prove the effect of preservation. The improved cryopreservation of cornea with glycerol may be a convenient, economic and effective method for eyebank.