目的：揭示眼科局部麻醉剂盐酸奥布卡因（oxybuprocaine hydrochloride,OBPC-HCl）对体外培养人角膜内皮（HCE）细胞的影响作用,为眼科临床安全用药提供实验依据。方法：用不同浓度OBPC-HCl处理体外培养的HCE细胞,在倒置显微镜下观察细胞的生长和形态变化,用吖啶橙/溴化乙锭（AO/EB）荧光双染色法检测质膜的通透性,用琼脂糖凝胶电泳法检测DNA的断片化,用透射电镜检测细胞的超微结构。结果：OBPC-HCl在62.5mg/L~4g/L的浓度范围内均能不同程度地引起HCE细胞出现细胞皱缩、胞内空泡化、质膜通透性增大、染色质凝缩、凋亡小体和DNA断片化等典型的细胞凋亡特征,并具有浓度和时间依赖性,临床使用浓度4g/L OBPC-HCl对HCE细胞的凋亡诱导作用最大,处理1h后HCE细胞的凋亡率已高达100%。结论：OBPC-HCl在62.5mg/L~4g/L的浓度范围内能显著诱导HCE细胞凋亡,在眼科临床应用中对HCE细胞的毒副作用极大。

AIM: To examine the effect of oxybuprocaine hydrochloride (OBPC-HCl) on in vitrocultured human corneal endothelial (HCE) cells and provide foundations for clinical safety use of OBPC-HCl.METHODS: After treated with OBPC-HCl at different concentrations, the growth situation and morphology of in vitrocultured HCE cells were checked under an inverted light microscope, membrane permeability was detected by acridine orange/ethidium bromide (AO/EB) double-fluorescent staining. DNA fragmentations and ultrastructure of HCE cells were observed by DNA agarose gel electrophoresis and transmission electron microscopy (TEM), respectively. RESULTS: In vitrocultured HCE cells treated with OBPC-HCl at a concentration between 62.5mg/L and 4g/L showed typical characteristics of apoptosis, including cell shrinkage, intercellular vacuolation, membrane permeability increasing, chromatin condensation, apoptotic body appearance and DNA fragmentation. The apoptosis-inducing effect of OBPC-HCl was in dose- and time-dependent manners. The greatest apoptosis-inducing effect of OBPC-HCl on HCE cells was found at the clinic concentration of 4g/L, and the apoptotic rate of HCE cells reached 100% after treated for 1 hour. CONCLUSION: OBPC-HCl at a concentration of 62.5mg/L-4g/L has an obvious apoptosis-inducing effect on HCE cells, and has huge toxic side effects on HCE cells during its clinic ophthalmic usage.

国家高技术研究发展863 计划(No. 2001AA625050)