脑脉络丛组织中锰毒性差异表达蛋白的体外验证  

Verification of manganese-related choroid plexus differentially expressed proteins in vitro

作　　者：董一文[1,2] 刘君丽[1,2] 刘羽[1,2] 敬海明[1,2] 赵超英[1,2] 马玲[1,2] 谭壮生[1,2] 李煜[1,2] 李芳[1,2] 李明[2] 焦智浩[2] 李国君[1,2] 

机构地区：[1]首都医科大学公共卫生与家庭医学学院,北京100069 [2]北京市疾病预防控制中心/北京市预防医学研究中心卫生毒理所,北京市食物中毒诊断溯源技术重点实验室

出　　处：《毒理学杂志》2013年第1期27-33,34,共8页Journal of Toxicology

基　　金：国家自然科学基金(81273108);首都卫生发展科研专项(首发2011-1013-03);北京市卫生系统高层次卫生技术人才培养项目(2011)

摘　　要：目的本实验室前期研究在锰染毒体内实验模型的大鼠脑脉络丛组织中鉴定到32个锰毒性相关的差异蛋白。本试验挑选出其中7个差异表达蛋白,包括抑制素蛋白(PHB1)电压阴离子通道(VDAC)、肌动蛋白β亚型(β-Actin)、应激性磷蛋白1(STI1)、热休克蛋白70(HSP70)、转甲状腺素蛋白(TTR)和中间丝波形蛋白(Vimentin),在大鼠永生化脉络丛上皮Z310细胞体外染锰模型中,对其蛋白和mRNA水平的变化趋势进行验证。方法氯化锰(0、50、100和200μmol/l)染毒Z310细胞24 h,采用蛋白印迹法(Western Blot)测定7个锰毒性相关蛋白的蛋白表达水平;同样剂量的氯化锰染毒Z310细胞12 h,以实时定量逆转录聚合酶链式反应法(Real time-RT PCR)测定7个蛋白在mRNA水平的表达情况。结果随着剂量的增加PHB1、β-Actin和STIP1在蛋白表达与mRNA水平均表现为上调的效应趋势,VDAC、HSP70、TTR和Vimentin在蛋白表达与mRNA水平均表现为下调的效应趋势;PHB1、β-actin、STIP1和TTR在Z310细胞中的表达效应趋势与体内动物模型脑脉络丛中锰毒性差异蛋白的变化趋势相符,而VDAC、HSP70和Vimentin在Z310细胞中的表达效应趋势则与之相反。结论氯化锰对PHB1、β-actin、STIP1和TTR的毒性效应在体内和体外是一致的,染锰后其蛋白表达和mRNA水平的变化趋势是可靠准确的。这为开展锰致脑脉络丛组织及其上皮细胞的毒性效应及其分子机制研究提供了有价值的线索。Objective In our previous study,a total of 32 Mn-related differentially expressed proteins were identified by 2D-PAGE combined with Nano-LC-MS/MS in an in vivo Mn-toxicity rat model.This study aims to further verify the 7 selected proteins（PHB1,VDAC,β-actin,HSP70,STIP1,TTR,Vimentin） at transcriptional and translational level respectively in immortalized choroidal epithelial Z310 cells in vitro under manganese chloride（MnCl2） exposure.Methods The expressed level of 7 proteins and their mRNA were detected by Western Blot and Real Time RT-PCR,following MnCl2（0,50,100,200 μmol/l） exposure for 24 or 12 h in Z310 cells.Results The results demonstrated that PHB1,β-actin and STI1 were up-regulated and TTR was down-regulated at both transcriptional and translational levels as compared to controls,which are in accordant with results of in vivo study.Whereas VDAC,HSP70 and Vimentin were down-regulated at both transcriptional and translational levels as compared to controls,which are opposite to the results of in vivo study.Conclusion Taken together,this study validated that Mn toxic effects on PHB1,β-actin,STIP1 and TTR in CP are accurate and reliable,which provide valuable clues for elucidating the molecular mechanism of Mn toxicity on choroid plexus epithelial cells.

关 键 词：锰毒性差异表达蛋白 脉络丛 永生化Z310细胞 血-脑脊液屏障 蛋白印迹 实时定量逆转录聚合酶链式反应 

分 类 号：R994.6[医药卫生—毒理学]