利多卡因对人角膜上皮细胞的毒性作用及其机理研究

发布时间：2018-11-19 10:22

【摘要】：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1.25~10.00 g/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0.625~10.000 g/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;Annexin V/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0.625 g/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。
[Abstract]:Human corneal epithelial (HCEP) cells were treated with lidocaine (lidocaine) at different concentrations. The cells were observed by light microscopy, MTT, fluorescence staining, DNA electrophoresis, TUNEL,. The toxic effect of lidocaine on HCEP cells and its mechanism were studied by flow cytometry and transmission electron microscopy. The results of light microscopy and MTT showed that lidocaine at 1.25g / L of 10.00 g / L had a significant toxic effect on HCEP cells in a dose-and time-dependent manner. The results of AO/EB fluorescence double staining showed that lidocaine (0.625 渭 g / L) could significantly increase the plasma membrane permeability of HCEP cells, and the apoptosis rate was also concentration-dependent and time-dependent. DNA electrophoresis and TUNEL analysis showed that lidocaine could induce DNA fragmentation in HCEP cells. The results of TEM showed that lidocaine could induce the ultrastructure of HCEP cells, such as cytoplasmic vacuolation, chromatin concentration, mitochondria swelling and structural disorder of crest, apoptotic corpuscles, etc. The results of flow cytometry with Annexin V/PI staining showed that lidocaine could induce the evagination of phosphatidyl serine (PS) in the cytoplasmic membrane of HCEP. The results of ELISA showed that lidocaine could also increase the expression of cysteinase-3-8 and 9-10 in HCEP cells, suggesting that lidocaine could induce apoptosis rather than necrosis of HCEP cells. It can be seen that lidocaine has significant cytotoxicity to HCEP cells when the mass concentration is greater than 0.625 g / L, and has a concentration-and time-dependent effect, and its toxic effect is achieved by inducing apoptosis. It has great toxicity and side effects in clinical application of ophthalmology, and should be used with caution.
【作者单位】：中国海洋大学海洋生命学院角膜组织工程实验室;
【基金】：国家高技术研究发展计划(“八六三”计划)资助项目(2006AA02A132)
【分类号】：R77

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