大鼠脉络丛组织中锰毒性差异蛋白的筛选、鉴定和GO分析  

Screening,identification and GO annotated analysis of manganese-related differentially expressed proteins in the choroid plexus of rats

作　　者：敬海明[1,2] 杨帆[3,4] 刘建中[2] 高文晖[2] 张拓[4] 赵超英[1,2] 马玲[1,2] 郑珊[1,2] 聂燕敏[1,2] 杜宏举[1,2] 张馨月[4] 原剑[3,4] 潘晨松 蒲海 李静[1] 魏开华[3,4] 李国君[1,2] 

机构地区：[1]北京市疾病预防控制中心卫生毒理所,北京100013 [2]首都医科大学公共卫生与家庭医学学院 [3]军事医学科学院放射与辐射医学研究所 [4]北京蛋白质组研究中心蛋白质组学国家重点实验室 [5]美国布鲁克.道尔顿公司北京代表处

出　　处：《毒理学杂志》2011年第1期25-29,共5页Journal of Toxicology

基　　金：北京市留学人员科技活动择优资助项目(No.2007-62);2007市属公益院所改革与发展项目(No.2007);北京市"十百千"卫生人才培养专项经费资助("百"层次)(No.2007-29);北京市"十百千"卫生人才培养专项经费资助("十"层次)(No.2009-1);国家重大科技专项项目子课题(No.2008ZX10002-106)

摘　　要：目的应用光镜、透射电镜及差异蛋白质组学技术探讨锰对构成血-脑脊液屏障的脑脉络丛组织(choroidplexus,CP)的病理损伤作用及其差异表达蛋白,并对这些差异蛋白按其gene ontology(GO)注释进行细胞学组分、分子功能与生物学过程的分类分析。方法采用雄性SD大鼠(1.5月龄)进行腹腔注射无水氯化锰(6 mg Mn/kg B.W)建立3个病程(30、90 d及90 d后无处理观察30 d)的锰中毒动物模型,分离其双侧侧脑室CP后,应用光镜与透射电镜观察锰所引起的CP病理形态学改变,同时应用蛋白质组学技术筛检出锰毒性相关的差异表达蛋白。结果病理形态学观察发现锰可引起CP上皮细胞发生不规则萎缩变小,微绒毛结构紊乱、缩短,胞浆内出现空泡,核质凝聚,线粒体结构破坏,细胞之间的紧密连接出现部分断裂或消失等,并且这些改变随病程发展表现为加重的趋势;2D-PAGE结合nano-LC-MS/MS的方法鉴定到了32个锰病程相关的差异蛋白质,其中有27个上调蛋白,5个下调蛋白。GO分类分析后发现差异表达蛋白主要分布在线粒体、膜表面以及细胞质内,以结合、催化活性以及转运功能为主,参与代谢与转运等生物学过程。结论锰可引起CP的细胞及亚细胞结构发生病理性损伤并表现为一定的时效性,即便停止锰染毒上述损伤仍然进行性加重;同时,还可造成CP中特定功能蛋白质随病程进展发生上调或下调的变化,这些改变很可能是锰引起BCB结构损伤和功能失调的分子基础,其功能蛋白质组学的研究值得进一步深入开展。Objective This study was designed to investigate if sub-chronic manganese（Mn） exposure led to changes of protein expression and structural damage in the choroid plexus（CP）,a brain tissue critical to material transport by the blood-CSF barrier（BCB）.Mn-toxicity-related differential expressed proteins in the CP were further analyzed according to Gene Ontology（GO） annotation by cellular distribution,molecular function and biological process.Method Male rats（1.5 months old） were received daily ip injection of either MnCl2（6 mg Mn /kg） or saline（as controls） for 30,90 or 90 days allowed for additional 30-day convalescence.The CP tissues in lateral ventricles were collected at each time point.Result Light microscopy and transmission electron microscopy revealed that Mn exposure resulted in flattened and shrunken cell layer,cytoplasmic vacuolation,nuclei and chromosome condensation,mitochondrion destruction,microvilli shortening,and partial disconnection in intracellular junctions between two adjacent epithelial cells.The structural alteration was mild after 30-day exposure and moderate in 90-day animals.Noticeably,the intracellular damage after 30-day convalescence was even worse than that at 90 days,suggesting a long lasting pathological damage even after Mn exposure was ceased.A total of 32 differential proteins were indentified by ZD-PAGE combined with Nano-LC-MS /MS,of which 27 were up-regulated,5 were down-regulated.Based on the information of GO categories,these differentially expressed proteins mainly locate in the mitochondria,membrane surface and cytoplasm,involving in the function of binding,catalytic activity and transportation and playing a critical role in the biological processes of metabolism and transport.Further verification and analysis are in progress.Conclusion Taken together,these data suggest that Mn not only damages the structural integrity of CP,but also alters the expressions of proteins critical to BCB function.

关 键 词：脉络丛 血-脑脊液屏障 锰 差异表达蛋白 GO分析 

分 类 号：R994.6[医药卫生—毒理学]