利多卡因对人角膜上皮细胞的毒性作用及其机理研究

利用不同浓度利多卡因（ｌｉｄｏｃａｉｎｅ）处理体外培养的人角膜上皮（ＨＣＥＰ）细胞系细胞，利用光镜观察、ＭＴＴ、荧光染色、ＤＮＡ电泳、ＴＵＮＥＬ、流式细胞仪和透射电镜方法研究了利多卡因对 ＨＣＥＰ细胞的毒性作用及其机理。光镜观察和 ＭＴＴ检测结果显示，质量浓度 １２５～１０００ｇ／Ｌ的利多卡因对 ＨＣＥＰ细胞具有显著的毒性作用，并具有浓度和时间依赖性；ＡＯ／ＥＢ荧光双染色结果显示，质量浓度 ０６２５～１００００ｇ／Ｌ的利多卡因可引起 ＨＣＥＰ细胞的质膜通透性显著提高，细胞凋亡率也具有浓度和时间依赖性；ＤＮＡ电泳和 ＴＵＮＥＬ检测结果显示，利多卡因能引起 ＨＣＥＰ细胞发生 ＤＮＡ断片化；ＴＥＭ观察结果显示，利多卡因能引起 ＨＣＥＰ细胞的超微结构出现了凋亡细胞的形态结构特征，如胞质空泡化、染色质浓缩、线粒体膨胀且嵴的结构紊乱、出现凋亡小体等；ＡｎｎｅｘｉｎＶ／ＰＩ染色的流式细胞仪检测结果显示，利多卡因能引起 ＨＣＥＰ细胞质膜中的磷脂酰丝氨酸（ＰＳ）发生外翻变化；ＥＬＩＳＡ检测结果显示，利多卡因还能引起 ＨＣＥＰ细胞中胱冬肽酶3、8、9、10表达量的增加，表明利多卡因确能引起 ＨＣＥＰ细胞发生细胞凋亡，而不是细胞坏死。由此可见，利多卡因在质量浓度大于 0.625ｇ／Ｌ时对 ＨＣＥＰ细胞具有显著的细胞毒性，并具有浓度和时间依赖性，且其毒性作用的发挥是通过诱导细胞凋亡实现的，在眼科临床应用中具有很大的毒副作用，应谨慎使用。

Cytotoxic effects of lidocaine on human corneal epithelial cells and its underlying mechanisms

To investigate the cytotoxic effect of lidocaine on human corneal epithelial （HCEP） cells and its underlying mechanism, in vitro cultured HCEP cells were treated with lidocaine at different doses and examined by light microsco- py, MTT assay, acridine orange/ethidium bromide （AO/EB） double-fluorescent staining, DNA electrophoresis, TUNEL assay, flow cytometry, and transmission electron microscopy （TEM）. Results of light microscopy and MTT assay showed that lidocaine at doses of 1.25 N 10.00 g/L exhibited significant cytotoxicity to HCEP cells, in a dose-and time-dependent manner. Results of AO/EB double-fluorescent staining showed that lidocaine at doses of 0. 625 - 10. 000 g/L elevated the plasma membrane permeability of HCEP cells, and the apoptotic rate of lidocaine-induced HCEP cells was also in a dose-and time-dependent manner. Results of DNA electrophoresis and TUNEL assay showed that lidocaine induced DNA fragmentation of HCEP ceils. TEM observation showed that lidocaine induced ultrastructural changes of HCEP cells which were similar to that of apoptotic cells, such as cytoplasmic vacuolation, chromatin con-densation, disordered cristae in swollen mitochondria, presence of apoptotic bodies, etc. Flow cytometry of annexin V/ PI staining showed that lidocaine induced translocation of phospholipid phosphatidylserine （PS） in plasma membranes of HCEP cells. ELISA assay showed that lidocaine induced over-expression of caspase -3,-8 ,-9,-10 in HCEP cells, indi- cating that lidocaine could induce apoptosis of HCEP cells, not necrosis. In conclusion, lidocaine at dose above 0. 625 g/L has significant cytotoxicity to HCEP cells in dose-and time-dependent manners, which is realized by inducing apoptosis in these cells, suggesting the inescapable sever cytotoxic side effect of lidocaine in eye clinic which should be utilized with attention.