基于金属组学的铅铜相互作用在铅致神经损伤中机制研究

The level of the metal ion and metal protein are proved to be closely related to the pathological state, or even a key point. Our preliminary studies showed that lead exposure resulted in the change of copper, copper transport proteins and two copper binding proteins in blood and hippocampus. Therefore the current project aimed to further explore the mechanism of lead induced neurotoxicity based on the concept of metallomics including copper distribution in tissues and cells as well as differential copper binding copper. In this study, laser capture micro cutting system united LCP-MS were applied to track the copper distribution spectrum or change of copper binding proteins in neurons or glia cells of rats following lead exposure. IMAC-Cu separation techniques, mass spectrometry and gene ontology (GO) analysis were used to indentify identification copper binding proteins and then annotated and classification based on their function following lead exposure. Meanwhile those differential copper binding proteins were verified in experimental animals, lead-exposed workers and lead poisoning patients. Moreover The copper change mechanism were also explored by applying the approaches of molecular biology technique At last, clioquinol, the copper compelling agent, were administrated to lead exposed rats in order to conduct prevention or treatment study of lead poisoning. In a word, the implementation of this project not only provide theory basis for mechanism of lead exposure induced neurotoxicity and screening effective biomarkers. Moreover our further finding will make new data for exploring the mechanism of other heavy metal toxicity.

金属离子水平和金属蛋白的表达与机体病理状态密切相关，甚至是关键环节。申请者的前期研究显示铅暴露可导致血清、脉络丛和海马中铜、铜转运蛋白和两种铜结合蛋白发生改变，因此本项目从金属组学的角度，进一步探讨铅和铜相互作用在铅致神经损伤的机制，即铜元素分布谱改变和差异铜结合蛋白的筛选。项目通过建立铅暴露的时间效应模型，联合显微激光切割系统、ICP-MS技术和分子生物学手段探讨不同组织、不同细胞中铜和铜结合蛋白表达的变化及其机制；联合IMAC-Cu分离技术、质谱分析和基因本体论分析方法对铅暴露后差异铜结合蛋白进行分离、鉴定和分类，然后对筛选出的差异蛋白在实验动物、铅作业工人和铅中毒病人中进行验证。此外应用铜的络合剂氯碘羟喹进行铅神经毒性的防治研究。通过对铅暴露后铜元素分布的改变和差异铜结合蛋白的探讨，为进一步探讨铅致神经损伤的机制和标志物研究提供了依据，同时也为其它金属神经毒性机制的探讨提供重要信息。

金属元素及金属相关蛋白组成的金属组在神经退行性疾病起着重要作用，而有关铜金属组在铅神经毒性中的作用未见报道。元素组研究表明铅暴露大鼠海马、皮质、脉络丛和脑脊液中主要变化的元素有Cu，Fe和Na。并绘制了铅暴露后不同脑区和不同屏障中Pb、Cu，Fe 的变化图谱。铅暴露后神经元和神经胶质细胞铜稳态失调，表现为铜含量增加、铜转运蛋白CTR1、ATP7A功能异常。同时，铅暴露大鼠的血脑脊液屏障维持中枢神经系统铜稳态功能受损，脉络丛中铜相关蛋白表达改变。蛋白质组结果显示，铅暴露大鼠海马差异蛋白包括铜转运蛋白、伴侣蛋白和具有铜结合域的GSTm1、HIF1、COX1、MT等，其中GSTm1、COX1、MT可作为铅作业工人神经损伤的候选标志物。铅暴露导致仔鼠中枢神经系统的铁稳态失调，表现为脑组织中铁含量增加、铁转出蛋白FPN1及其辅助蛋白HP和CP表达下降。铜络合剂氯碘羟喹干预可缓解铅暴露导致的运动神经损伤和学习记忆能力下降。总之，铅暴露可导致中枢神经系统铜稳态失调以及血脑脊液屏障维持中枢神经细胞铜稳态功能受损，这可能与铜转运蛋白、具有铜结合域的氧化损伤蛋白功能失调有关。提示这些金属相关蛋白可作为铅神经损伤的候选标志物和治疗的靶点。本研究结果也为其他二价金属锰、铜金属神经损伤的研究提供了新的思路。

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