CAJ | 학술논문

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。
이용불동농도리다잡인（ lidocaine）처리체외배양적인각막상피（ HCEP）세포계세포，이용광경관찰、MTT、형광염색、DNA전영、TUNEL、류식세포의화투사전경방법연구료리다잡인대HCEP세포적독성작용급기궤리。광경관찰화MTT검측결과현시，질량농도1.25～10.00 g/L적리다잡인대HCEP세포구유현저적독성작용，병구유농도화시간의뢰성；AO/EB형광쌍염색결과현시，질량농도0.625～10.000 g/L적리다잡인가인기HCEP세포적질막통투성현저제고，세포조망솔야구유농도화시간의뢰성；DNA전영화TUNEL검측결과현시，리다잡인능인기HCEP세포발생DNA단편화；TEM관찰결과현시，리다잡인능인기HCEP세포적초미결구출현료조망세포적형태결구특정，여포질공포화、염색질농축、선립체팽창차척적결구문란、출현조망소체등；Annexin V/PI염색적류식세포의검측결과현시，리다잡인능인기HCEP세포질막중적린지선사안산（ PS）발생외번변화；ELISA검측결과현시，리다잡인환능인기HCEP세포중광동태매-3、-8、-9、-10표체량적증가，표명리다잡인학능인기HCEP세포발생세포조망，이불시세포배사。유차가견，리다잡인재질량농도대우0.625 g/L시대HCEP세포구유현저적세포독성，병구유농도화시간의뢰성，차기독성작용적발휘시통과유도세포조망실현적，재안과림상응용중구유흔대적독부작용，응근신사용。
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 1.25~10.00 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 cells.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.