无血清器官培养法保存大鼠角膜内皮细胞的活性

目的:研究两种新型培养基-内皮细胞培养基(endothelial cell medium,ECM)和无动物成分培养基(animal compoundfree medium,ACF)在无血清器官培养法保存角膜内皮细胞活性方面的应用效果。方法:将大鼠角膜密闭保存于ECM和ACF培养基中4wk,再经葡聚糖T500脱水2d。以含低浓度牛血清的MEM基础培养基作为对照。计数保存前后角膜内皮细胞密度,检测保存结束大鼠角膜内皮层中胞质紧密粘连蛋白1(zonula occludens-1,ZO-1)的表达水平,评估内皮细胞层的屏障功能。结果:保存结束后,MEM+20mL/L FBS对照组大鼠的角膜内皮细胞密度值最低,ECM+20mL/L FBS组则高达2250±202个/mm2,ECM和ACF组结果与ECM+20mL/LFBS组接近,细胞死亡率也较低,组间差异有统计学意义(F=28.965,P=0.000)。冰冻切片显示ZO-1在保存后大鼠角膜内皮层成功表达。RT-PCR检测表明ZO-1mRNA在各组的表达趋势与内皮细胞密度结果一致(F=592.751,P=0.000)。结论:无血清ECM和ACF培养基可以很好地保持器官培养法保存角膜内皮细胞的活性和紧密连接的屏障功能。

Effect of serum free organ culture on corneal endothelial cells viability

·AIM:To investigate the effect of serum free organ culture on corneal endothelial cells viability by comparing two specific endothelial cell media—endothelial cell medium(ECM)and animal compound free medium(ACF)with the conventionally used basic minimum essential mediun(MEM).·METHODS:Rat corneas were cultured in ECM and ACF test media with or without 20mL/L fetal bovine serum(FBS)for 4 weeks and then dehydrated in dextran T500 for 48 hours.The viability evaluation parameters of corneal endothelial cells included:endothelial cell density before and after preservation;The immunohistochemistry and semi-quantitative expression of zonula occludens-1(ZO-1)using RT-PCR in rat corneas whose epithelium had been predissected.·RESULTS:After cultivation,the grafts cultured in MEM+20mL/L FBS control group exhibited the lowest endothelial cells density(1875±162 cells/mm2).The highest mean corneal endothelial cell density was found in ECM+20mL/L FBS medium group(2250±202 cells/mm2)and the corneas cultured in ECM and ACF media achieved similar endothelial cells loss(F=28.965,P=0.000).Vital staining revealed the lower numbers of damaged corneal endothelial cells in ECM(with or without FBS)and ACF groups.ZO-1 could be seen in corneal endothelia of frozen sections.ZO-1mRNA expression in rat corneas without epitheliums exhibited the lowest level in MEM medium and the highest level in ECM(with or without FBS)and ACF media(F=592.751,P=0.000).·CONCLUSION:Serum-free ECM and ACF media can preserve rat corneal endothelial cells viability and tight junction integrity better than conventional MEM medium with low FBS.They show great superiority and may play an important part in serum free organ culture corneas in the future.·