基于分子暴露组学的铬致癌早期效应研究

The carcinogenesis of chromium results from gene-environment interaction.Molecular systems biology provides new platform to investigate early carcinogenesis of Chromium in human. To obtain critical molecular biomarkers for molecular exposome, the current study is planning to screen a set of differentially expressed proteins and circulatory miRNA in serum and differentially methylated genes in peripheral lymphocytes from the blood specimens which have been collected from a previous sectional study. Two-Dimensional Electrophoresis-Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry will be used to screen differentially expressed proteins, which are then quantified through enzyme linked immunosorbent assay. miRNA chips will be adapted to screen differentially expressed circulatory miRNA, and real time PCR will be utilized to quantify them. Methylation-specific PCR technology will be carried out to screen and quantify differentially methylated genes. These identified molecular biomarkers will be validated by a cohort study with exposed individuals from a chromate manufacture in Shandong province and nearby controls without chromate exposure. Environmental monitoring and questionnaire will be meanwhile carried out.Simultaneously Cytokinesis-Block Micronucleus Assay (CBMN), Sister Chromatid Exchange Analysis (SCE) and serum 8-hydroxy-2'-deoxyguanosine assay (8-OHdG) will be measured to assess chromosome and DNA damage levels. The information will be integrated into a dynamic Bayesian network as variables to mimic environmental chromium-gene-early effect interactions. The pivotal variables in the network with close relationship to chromium exposure and chromosome or DNA damage level will be considered as biomarkers of exposure or early effect to predict early effects during carcinogenesis.

铬致癌作用是环境和机体交互作用的结果，系统分子生物学为铬早期致癌过程研究提供了新的手段。本研究利用流行病学横断面调查结合健康体检，获得符合入选标准的铬盐接触者和非接触者血样，用二维电泳串联基质辅助激光解析电离-时间飞行质谱筛选血清差异表达蛋白并通过酶联免疫法定量；用miRNA芯片筛选差异循环miRNA并进行PCR定量；利用甲基化特异性PCR筛选差异甲基化基因并定量。进一步通过流行病学队列研究，验证上述差异大分子随铬接触时间和接触水平的变化规律，结合环境暴露、生物暴露水平和问卷调查信息，对铬接触人群进行早期分子暴露组学分析；同时借助胞质分裂阻滞微核试验、姐妹染色单体交换试验、血清8-羟基脱氧鸟嘌呤水平，分别评价机体染色体和DNA损伤程度；综合以上数据，建立贝叶斯网络模型，模拟并分析铬的环境-基因-早期效应相互作用网络，以其中关键变量作为铬接触和早期效应标志物，预测铬致癌早期效应及可能风险。

建立铬酸盐接触人群队列，获得符合入选标准的血样、尿样和空气滤膜。利用ICP-MS、单细胞凝胶电泳、胞质阻滞微核和ELISA测定了全血和滤膜铬的含量、DNA和染色体损伤及血清8-羟基脱氧鸟嘌呤含量；利用液质联用型质谱仪、miRNA芯片和时间飞行质谱等分别对铬酸盐接触者差异表达血清蛋白谱、血浆miRNA表达谱、全基因组甲基化、羟甲基化及DNA损伤修复过程中关键基因CpG岛甲基化水平进行了鉴别和分析；扩大人群样本量，结合体外细胞实验对上述差异表达或修饰分子进行了验证。利用动态贝叶斯模型和生物信息学方法，对铬酸盐接触致差异表达的上述分子进行了功能和交互网络分析。研究发现：①接触者外周全血铬水平不仅与空气铬浓度相关(R=0.38, P<0.001)。还可反映机体近1.5年内的累积暴露量。接触者在现有铬酸盐职业接触限值(50μg/m3)水平下仍可观察到肺损伤、遗传损伤及相关因子的改变；②铬酸盐接触改变的44种血清蛋白主要与机体免疫调节相关。血清C反应蛋白、音猬因子含量分别与全血铬呈负相关(R=-0.363, P=0.003)和正相关（R=0.526，P=0.007），有望作为铬酸盐接触血清早期效应标志物；③铬酸盐接触可引起外周血全基因组低甲基化和DNA修复关键基因多CpG岛高甲基化的改变。MGMT、HOGG1和RAD51的CpG岛甲基化水平与全血铬及遗传损伤之间呈正相关，与其相应基因的表达量呈负相关，可作为铬酸盐接触表观遗传效应标志物；④铬酸盐接触可引起6种miRNA表达改变，miR-3940-5p表达与全血铬存在暴露-反应关系(y=26.3ln(Cr))。铬可拮抗锌-金属反应转录因子调节miR-3940p宿主基因KHSRP抑制miR-3940-5p表达。进而低表达的miR-3940-5p负反馈调节XRCC2表达增加，促进同源重组修复，从而在铬酸盐遗传损伤起保护作用。本研究为铬酸盐接触提供了一组新的早期效应标志物，初步明确了miRNA、甲基化修饰在铬酸盐致遗传损伤中的作用，提出了更安全的全血和空气铬职业接触限值，为预测六价铬早期致癌效应及健康风险评估提供了可能，为落实铬酸盐接触的一级预防提供了技术支撑。培养研究生10名，含博士6名；已发表论文21篇，其中SCI收录11篇。特邀报告1次，分会场报告5次。