全氟有机物干扰脂类代谢的分子机制及暴露标志物研究

Perfluoroalkyl chemicals (PFCs) are stable man-made compounds with many industrial and commercial uses. Many reports have indicated that PFCs are widespread in the sera of diverse general human populations, as well as in the environment and in wildlife. The ubiquitous presence and persistence of PFCs in the environment and within the human body have led to efforts to understand the toxicological hazards that may be associated with exposure..MicroRNAs (miRNAs) are endogenous small non-coding RNAs. They play an important role in regulating gene expression by blocking translation or trigger the degradation of target mRNAs. Recently, studies showed that serum contained a large amount of miRNAs and the pattern of serum miRNA was altered in various disease conditions, which could potentially be used as novel noninvasive biomarkers for diagnosis and therapy. Although preliminary, our study found that several circulating miRNAs were differentially expressed in occupational flurochemical workers samples compared with general population samples. The result implied that the high concentration of PFOA in occupational workers potentially arouses physiological responses and leads to an alteration in the level of circulating miRNAs. We hypothesized that the levels of specific circulating miRNAs may be used as biomarkers to monitor potential pathological injuries by serum PFCs..Animal studies suggest that the liver is a primary target organ for perfluoroalkyl acid physiological activity. The structural resemblance of PFCs to natural fatty acids has raised concern that PFCs may exert deleterious effects, especially on lipid regulation. PFCs can act as a ligand for peroxisome proliferator activated receptors (PPARs) and lead to beta-oxidation of fatty acids. However, PPAR-agonistic mode can not explain all of the hepatic toxicity, and PFCs may result in lipid disturbance by other lipid-regulated transcription factors..In our present program, firstly, we will detect the concentration of serum PFCs in PFC exposed animals using high-pressure liquid chromatography/tandem mass spectrometry (HPLC-MS/MS), detect the circulating miRNA levels using miRNA array and qPCR assay, and identify the relationship between the chemical and serum miRNA levels. By these methods, we hope that we find the serum miRNAs which can be used as biomarker for PFC contamination. The second aim of our program is to identify the lipid-regulated transcription factors which can be activated by PFCs exposure, to identify the alteration of key genes involved in the catabolism, transport, and anabolism of lipids, to detect the alteration in hepatic miRNAs using TaqMan qPCR assay, and identify their targets using iTRAQ labeling and MS/MS method, to interrogate the relationship between transcription factors and miRNAs, the two kinds of key regulators for gene transcription and expression, and finally identify the mechanism behind the lipid disturbance of PFCs.

全氟有机物(PFC)是一类新型持久性有机污染物。PFC在全球环境介质中被广泛检出，且具有多种生物毒性。目前，PFC干扰肝脏脂类代谢的分子机制尚未阐明，缺乏能表征PFC污染的标志物。我们的初步结果显示血清微小RNA(miRNA)对PFC暴露反应敏感，值得深入研究。本项目拟通过PFC暴露小鼠，采用HPLC-MS/MS确定PFC在血清和肝脏中的分布模式；通过研究PFC与血清miRNA的剂量（时间）-效应关系，找出基于血清miRNA的敏感、有效的暴露标志物。研究PFC暴露后调节肝脏脂类代谢的关键转录因子(TF)及其下游基因和肝脏miRNA的变化；利用同位素标记定量(iTRAQ)与质谱技术研究miRNA调控的靶基因；利用荧光素酶报告基因等多种实验确定TF与miRNA的相互关系，从而最终确定PFC干扰肝脏脂代谢的分子机制，为评估PFC对环境健康的影响和制定PFC管理策略提供科学依据。

全氟烷酸化合物因其独特的物化性质被广泛用于工业和商业产品中，但其可能具有多种毒性效应。(1)我们研究了PFOA暴露小鼠后血清循环miRNAs表达的变化，发现在1.25 mg/kg/d和5 mg/kg/d剂量处理组分别有24和73个miRNAs发生显著改变；PFOS暴露小鼠血清也呈现类似的miRNA表达谱改变。该结果表明循环miRNAs可能与PFOA的生物效应有关。(2) PFOA暴露能刺激SREBP蛋白成熟，并阻止其前体转运蛋白INSIG1和INSIG2，以及F-box和WD-40结构域蛋白7等在暴露动物肝脏中的表达。介导SREBPs成熟的miR-183-96-182簇在PFOA暴露后显著升高。(3) PFOA暴露28天能改变肝脏PI3K-AKT信号通路，影响糖代谢并诱导小鼠胰岛素高敏。(4) PFOA暴露导致小鼠肝脏ER应激，导致未折叠蛋白反应(UPR)，其在PFOA的肝脏毒性中发挥作用，而化学分子伴侣4-PBA能减轻此毒性效应。(5) HL-7702细胞暴露于50 μM PFOA 48 h和96 h后，差异蛋白中有多达46种与细胞增殖与凋亡相关。我们发现低剂量PFOA (50–100 μM)暴露能通过加速细胞由G1期进入S期而促进细胞增殖，而高剂量PFOA (200–400 μM) 暴露主要使细胞阻滞在G0/G1期。(6) PFDoA暴露导致肝脏ROS水平增高，使用PPARα RNAi，PPARα拮抗剂GW6471和激动剂WY14643对离体大鼠原代细胞的研究表明PPARα在抑制PFDoA所致氧化应激中发挥保护作用。(7) 我们使用iTRAQ方法鉴定了PFNA暴露小鼠后肝脏差异蛋白，发现PFNA暴露不但导致脂肪酸氧化效应增强，而且也活化脂肪酸和胆固醇合成信号通路。另外研究了PFNA处理所致肝脏miRNAs谱改变，发现miRNAs通过其转录后蛋白抑制效应在PFNA所致肝脏效应中发挥作用。