【作者】 严京； 祁明信； 黄秀榕；

【Author】 JingYA~1,Mingxin Qi~2,Xiurong HUANG~3 (1.Eye Hospital China Academy Of Chinese Medical Sciences Internist,Beijing,100040,China;2.The Second General Hospital,Fujian University of Traditional Chinese Medicine,Fuzhou,350003,China;Fujian University of Traditional Chinese Medicine,Fuzhou,350003,China)

【机构】 中国中医科学院眼科医院； 福建中医学院附属第二人民医院眼科； 福建中医学院病理生理研究中心；

【摘要】 目的:探讨阿魏酸钠(SF)防护紫外线(UV)照射诱导的人晶状体上皮细胞(HLEC)损伤的作用,寻找SF防护细胞损伤的作用机制。研究方法本研究采用SF与HLEC共同孵育,再用UV照射HLEC后,通过流式细胞仪检测,RT-PCR、western等方法探讨SF抗氧化损伤和细胞凋亡作用,并采用蛋白质组学寻找差异蛋白。研究结果:UV照射可降低细胞活性,诱发细胞凋亡和坏死。SF可提高UV照射后的HLEC细胞活性,流式细胞仪检测SF组HLEC核内的DNA含量较UV组增加,线粒体跨膜电位较UV组升高(P<0.0 1)。SF组与UV组比较HLEC内SOD)、GSH-px,CAT含量及mRNA表达均升高(P<0.01)。蛋白质组学研究比较SF组与UV组得到:10个差异蛋白点;其中表达上调的差异点8个表达下调的差异点2个。并鉴定了其中3种蛋白。SF还可提高HLEC的波形蛋白表达。研究结论:SF可减轻UV照射引起的HLEC氧化损伤,提高HLEC抗氧化酶能力;SF减轻UV照射后引起HLEC凋亡的机制是通过提高HLEC核内DNA含量、降低细胞凋亡率以及通过升高线粒体跨膜电位、提高HLEC活性,减少HLEC凋亡和坏死。蛋白质组学方法鉴定出三种蛋白质参与SF防护UV照射损伤,这为进一步研究白内障发病机制提供新线索。更多还原

【Abstract】 Objective To investigation the inhibitory effect and relative Mechanisms of Sodium Ferulate(SF) protecting injury of human lens epithelia cells(HLEC) induced by ultraviolet(UV). This observation that using technologies of proteomics will provide a view in better understanding the mechanisms of UV-induced injury of human epithelia cells,and provides the experiment bases for clinical application of SF in the future.The experiments can be summarized as the following.Methods This work will use human lens epithelia B3 cells.HLEC were cultured and sub-cultured in vitro. The cultured HLEC were divided into control group;UV group;SF group and pirenoxine solution(PS) group.The cells of SF group and PS group were incubated with special concentration of,SF and PS respectively.And then,the cells were exposed under UV.After UV irradiation,the irradiated cells were further cultured for 4 hours.Effect of SF and PS was detected by methyl thiazolyl tetrazolium (MTT),optical microscope,transmission electron microscope,The effect of SF and PS on nucleus DNA content and the mean fluorescence intensity(MFI) of mitochondrial trans membrane potential was detected The activity and gene expression of superoxide dismutase(SOD),glutathione peroxidase (GSH-px) and catalase(CAT) were measured by chemical chromatometry and RT-PCR.With the proteomics approaches,using technology of two-dimensional electrophoresis(2-DE),gel dyeing and image analysis to obtaining different protein spots in various groups,and then using technology of Mass Spectrometry(MS) and bioinformatics to analysis and identifies protein spots,look for functional protein in HLEC induced by UV and target protein of SF preventing and treating injury of HLEC. The expression of vimentin that is one of proteins of cytoskeleton was determined by analysis of immunoblot to observer effect of SF on proteins of cytoskeleton.Results Effect of SF and PS on apoptosis of HLEC irradiated by UV.The result of MTT test show that UV group can reduce the opti- cal density values(OD) obviously compared to control group(P<0.01).The OD values of SF and PS group higher than that of UV group remarkably,and indicate dose-dependent within concentrations of SF and PS respectively.SF and PS show a better effect at the concentration of 100mg/L and 5mg/L respectively.The DNA content increased and the apoptosis rate decreased markedly in SF and PS group comparing with UV group.This change indicated that SF and PS all could inhibit apoptosis of HLEC induced by UV.Moreover,The DNA content of SF group is higher than that of PS group(P<0.01).The result of MFI of mitochondrial trans membrane potential show that UV group can reduce the MFI obvi- ously compared to control group(P<0.01).The MFI values of SF and PS group is higher than that of UV group remarkably.. Effect of SF and PS on oxidative damage of HLEC irradiated by UVThe contents and The expression of gene of SOD,GSH-px and CAT decreased in the UV group,however,the contents of three anti-oxidant enzymes increased markedly in SF and PS group compared with UV group.Effect of SF on proteomics of HLEC irradiated by UVUV group compared with control group:obtained different spots are 13,in which,5 spots regulated up.Four kinds of protein are identified by analysis of MS and bioinformatics.8 spots regulated down in 13 different spots.Three kinds of protein are identified.SF group compared with UV group:8 spots regulated up,in which,three kinds of protein are identified.Protein expression of vimentin down regulated in the damaged HLEC,that of SF and PS group up regulated greatly.Conclusion UV irradiation can induce apoptosis of HLECs,however,SF and PS have effects to abrogate apoptosis of HLEC induced by UV.Mechanisms of SF preventing and treating injury of Human Lens Epithelial Cell induced by UV through rising nucleus DNA content of HLEC,decreasing apoptosis rate,rising level of mitochondrial trans membrane potential and elevating activity of HLEC.UV radiation can induce oxidative damages of HLEC.SF and PS can safeguard these damages through rising content and mRNA expression of SOD,GSH-px and CAT.Proteome analysis of HLEC irradiated by UV show change of expression in many proteins.Proteins expressed down regulate are Vimentin,etl.,and proteins expressed up regulate are T-complex protein 1 subunit beta, heterogeneous nuclear ribonucleoprotein,etl.Proteomic mechanisms of SF preventing and treating injury of HLEC induced by UV through reversed expression of differential proteins.SF can regulate up expression of three kinds of protein that regulated down in UV irradiation.Western-blot analyses show expression of vimentin decrease in UV irradiation,however,expression of vimentin increased remarkably in SF.更多还原