外周血DNA甲基化差异性表达在铝职业暴露及铝暴露致认知功能损伤的作用研究

Aluminum is a chronic accumulation neuropoison, which can cause recognition functional disturbance such as the depression of learning memory abilities.Some reports showed that DNA methylation are associated with environmental pollutants exposure and the development of its related diseases.But there are few reports about the study of DNA methylation expression profile of peripheral blood in the occupational workers exposed to aluminum. So in this study we will explore the recognition function and the expression levels of DNA methylation in peripheral blood in the occupational workers exposed to aluminum in GuangXi mining district, and we will screen the differential DNA methylation of the promoter-specific of genome-wide of peripheral blood by using MeDIP assay, and verify the mRNA and methlation level expression of the differential methylated genes by RT-PCR and bisulfite genoimic sequencing. By analysing the relationship of the aluminum exposure,the recognition functional states and the differential expression of methylated genes we will decide the differential candidate genes which are associated with the cognitive impairment of occupational workers exposed to aluminium. We will treat the neuron cell line HCN-2 and human glial cells with AlCl3, and construct the cells of low or high expression of the candidate genes and treated with AlCl3,respectively, which will further test the differential of methylated genes.The results of this study will help to look for the biomarkers for the precaution of aluminum exposure and nervous system disease, and will support the theories for the early discovery and precaution of the recognition functional disturbance of occupational workers exposed aluminum, and will promote the development of epigenetic regulation mechanisms.

铝是一种慢性蓄积性神经毒物,可引起学习、记忆能力下降等认知功能障碍。研究表明，DNA甲基化与环境污染物的暴露及其相关的机体疾病的发生发展密切相关。而有关外周血DNA甲基化的变化模式在铝暴露、铝暴露引起认知功能的作用及其机制的研究未见相关报道。因此本研究拟通过在广西铝矿区选择铝暴露的职业人群，分析不同的铝暴露累积剂量下职业人群认知功能的变化，利用甲基化芯片技术筛选差异性甲基化基因并验证；综合分析铝暴露、认知功能与差异性表达的甲基化基因之间的关系以确定与铝暴露引发认知功能相关的差异性的目的甲基化基因；利用AlCl3对体外培养的人神经细胞HCN-2、人神经胶质细胞及对建立的目的甲基化基因低或高表达的细胞模型染毒，进一步验证差异性的目的甲基化基因的功能，寻找出预测铝暴露和神经系统疾病发生早期的分子标记物。本研究为职业铝暴露引起的认知功能障碍的早期发现及预防提供理论依据,有力促进表遗传机制。

本项目主要研究铝暴露的职业人群外周血基因组甲基化水平和人群认知功能的关系，获得了以下的研究结果：①利用AlCl3染毒，发现铝暴露可使大鼠认知功能下降，大鼠血清及脑组织的SOD活性降低，MDA水平升高。②进行了职业铝接触工人的职业性有害因素和认知功能调查，筛选出职业性铝暴露认知障碍的工人和非认知功能障碍的对照人群，发现职业性铝接触可以引起作业工人出现明显的心理状态、神经运动速度、准确性、协调能力及副交感神经调节功能的改变。③进行了职业流行病学调查，探讨铝职业暴露工人认知功能障碍检出情况。发现暴露组人群中有认知功能障碍者占10.70％，对照组人群中有认知功能障碍者占1.00％，差异有统计学意义（χ2＝25.57，P <0.001）。④采集上述两组人群外周血，抽提DNA和外周血淋巴细胞，联合采用高通量甲基化芯片和mRNA表达谱芯片技术筛选差异的甲基化基因。甲基化芯片结果发现Log2FC大于2的差异甲基化基因共42105个，其中下调25481个基因（60.52%），上调16625个基因（39.48%）。结合mRNA表达谱结果发现，甲基化和mRNA差异表达均大于2倍的基因共有688个，其中335个基因的甲基化表达上调而mRNA的表达下调（50.15%），333个基因的甲基化表达下调而mRNA的表达上调（48.40%）。而甲基化表达上调而mRNA表达下调的基因共5个，即PI3、SLPI、SNORD12、CA1、CHI3L1。⑤扩大采集上述两组人群外周血，提取外周血淋巴细胞RNA，采用实时荧光定量PCR对上述5个基因的mRNA表达进行检测。结果发现，发现SLPI、CA1、CHI3L1的表达量分别升高了约0.85倍、0.75倍、0.95倍。本研究为寻找出预测铝暴露和神经系统疾病发生早期的分子标记物，为职业铝暴露引起的认知功能障碍的早期发现及预防提供理论依据，有力促进表遗传机制的发展。