两种新型无血清培养基器官培养保存兔角膜内皮细胞活性的实验研究

目的研究两种新型培养基-内皮细胞培养基（ECM）和无动物成分培养基（ACF）在无血清器官培养法保存角膜内皮细胞活性方面的效果。方法将64枚兔角膜平均分为4份，分别密闭保存于ECM＋2％胎牛血清（FBS）、ECM和ACF等4种不同的培养基中4周，再以葡聚糖巧oo脱水2d。伊格尔最低基础培养基（MEM）作对照组。检测保存前后角膜内皮细胞密度、角膜厚度、内皮层中肌动蛋白微丝（F-actin）的表达和透射电镜下内皮细胞超微结构的变化。结果保存结束后，ECM＋2％FBS组的兔角膜内皮细胞密度最高（21234-240）个／mm^2，ECM和ACF组结果与其接近，内皮细胞死亡率也较低，对照组内皮细胞密度最低（1732±144）个／mm^2，3个实验组与对照组间差异有统计学意义（F=23．180，P=0．000）。免疫组织化学染色显示F-actin在兔角膜内皮层成功表达。WB半定量检测表明F-actin蛋白在各组角膜内皮层的表达趋势与内皮细胞密度计数结果一致，3个实验组与对照组间差异有统计学意义（F=159．876，P：0．000）。3个实验组内皮细胞的超微结构与正常角膜区别较小。结论在器官培养法保存过程中，无血清的ECM和ACF培养基可以较传统MEM培养基更好地保持角膜内皮细胞的活性和屏障功能，在无血清器官培养保存角膜方面具有应用前景。

Effect of two potential media on serum free organ culture of rabbit corneal endothelial cells

Objective To investigate two specific endothelial cell media (ECM and ACF) for their use in serum free organ culture of corneal endothelial cells comparing with the conventionally used basic MEM medium. Methods Rabbit comeas were cultured in ECM + 2％ FBS, ECM, ACF test media for 4 weeks and then dehydrated in 5％ dextran T500 for two days. The viability evaluation parameters of corneal endothelial cells included' endothelial cell density before and after preservation, the corneal mean central thickness before and after preservation,the immunohistochemistry and semi-quantitative expression of filament actin(F-actin) using western blotting in rabbit corneal endotheliums, and the ultrastructure observation of rabbit corneal endothelial cells under transmission electron micronscopy were determined. Results After cultivation, the highest rabbit mean corneal endothelial cell density was found in ECM + 2％ FBS medium group (2123 + 240)/mm2 and the corneas cultured in ECM and ACF media exhibited similar endothelial cells loss. The grafts cultured in MEM + 2％ FBS control group exhibited the lowest endothelial cells densities ( 1732 ± 144)/mm2 ( F = 23. 180, P = 0.000). Vital staining revealed the lower numbers of damaged corneal endothelial cells in ECM( with or without FBS) and ACF groups. F-actin could be seen in corneal endotheliums of frozen sections. F - actin protein expression in rabbit endotheliuns achieved the lowest level in MEM medium and the highest levels in ECM( with or without FBS) and ACF media( F = 159. 876, P = 0. 000).The ultrastructures of corneal endothelial cells of three test media remained good compared with normal cornea. Conclusion Serum-free ECM and ACF media showed superiority over conventional MEM medium with low FBS content in preserving corneal endothelial cells viability and tight junction integrity. They have great potentials and may play an important part in serum free organ culture corneas in the future.