节点文献
多环芳烃致肺癌预警标志物的代谢物靶标和代谢轮廓分析
Study of Forewarning Markers of Lung Cancer Causing by PAHs’ Using Metabolite Target and Profiling Analysis Technology
【作者】 康荣慧；
【导师】 王永生；
【作者基本信息】 南华大学 ， 劳动卫生与环境卫生学， 2010， 硕士
【摘要】 多环芳烃（polycyclic aromatic hydrocarbons,简称PAHs）是一类典型的环境持久性有机污染物,在职业高PAHs暴露环境下,容易诱发皮肤癌、肺癌等癌症,现已被多个国家列为优先控制的污染物。评价多环芳烃暴露水平可为环境健康风险预警评价和流行病学研究等提供依据。由于暴露途径以及影响因素复杂多样化,采用尿样中PAHs羟基代谢产物（主要有1-羟基芘、萘酚及菲、芴的代谢产物）作为生物标志物来综合评价人体暴露PAHs污染物的危害及肿瘤风险预警已逐渐成为研究的热点。因此建立PAHs羟基代谢物靶标分析新方法,对PAHs的羟基代谢产物进行代谢物靶标分析以及PAHs羟基代谢产物及其结构类似物的HPLC-FD高通量轮廓谱分析方法,并构建职业暴露PAHs人群的肺癌风险预测模型,对于PAHs所致肺癌预警、早期诊断、职业安全评价和肺癌的分子机制的深入研究有着重要的现实意义。本文第二章应用一阶导数同步荧光技术联用胶束增敏法建立了同时检测1-羟基芘、1-萘酚、2-萘酚、9-羟基菲和2-羟基芴的新方法。研究表明:在pH 5.0 B-R缓冲溶液中,加入吐温-20作为增敏剂,当选择激发单色器和发射单色器的波长差Δλ= 10 nm,只需一次扫描,利用计算机的导数功能得到一阶导数荧光光谱,可实现五组分的同时鉴别和定量测定。采用峰-零法分别在λ= 389.4 nm、332.4 nm、338.6 nm、355.8 nm、306.0 nm记录1-羟基芘、1-萘酚、2-萘酚、9-羟基菲和2-羟基芴导数荧光强度值。1-羟基芘、1-萘酚、2-萘酚,9-羟基菲和2-羟基芴的线性范围分别为1.75×10-9 4.50×10-6 mol·L-1 , 3.64×10-82.20×10-4 mol·L-1 , 8.18×10-91.20×10-4 mol·L-1 ,3.26×10-98.50×10-5 mol·L-1和4.88×10-95.50×10-6 mol·L-1 ,检出限分别为5.25×10-10 mol·L-1,1.10×10-8 mol·L-1,2.46×10-9 mol·L-1,9.77×10-10 mol·L-1和1.46×10-9 mol·L-1。本法快速、灵敏,可同时测定尿样中痕量1-羟基芘、1-萘酚、2-萘酚、9-羟基菲和2-羟基芴,结果与高效液相色谱法一致。本文第三章应用反相高效液相色谱-荧光-二级管阵列检测器串联建立了同时测定尿中5种多环芳烃和6种羟基多环芳烃的新方法。采用Shim-pack VP - ODS C18柱（150 mm×4.6 mm, 4. 6±0. 3μm）色谱柱分离,以甲醇/乙酸钠-乙酸缓冲液（pH 4.5）（65:35,v/v）为流动相,流速为1.0 mL·min-1,柱温为30°C;进样量为20μL。外标法定量,分离效果好,缩短了样品分析时间,30 min内可将11种待测物质完全分离。荧光检测器的检出限为0.0010.090μmol·L-1;二极管阵列检测器检出限为0.0500.900μmol·L-1;样本加标回收率为95.5%108.0%。已用于尿样测定,结果满意。本文第四章基于高效液相色谱法建立多环芳烃所致肺癌病人的预测模型。利用第三章建立起的反相高效液相色谱法检测得到36名肺癌病人和10名健康人尿中多环芳烃代谢轮廓谱,结合主成分分析法和偏最小二乘法对所得到的代谢轮廓谱数据进行分析,建立多环芳烃所致肺癌病人的预测模型,并初步筛选并鉴定出2个相对较为重要的具有显著异常改变的指标（1-萘酚,萘）,该法对多环芳烃所致肺癌的诊断有一定的参考意义。
【Abstract】 Polycyclic aromatic hydrocarbons （PAHs） are a large class of ubiquitous persistent organic pollutants, which may induce skin and lung cancers in occupational settings and have been listed in preferred controlling pollutants by many countries. The evaluation of exposure to PAHs can provide evidence for environmental health risk warning and epidemiological study. However, exposure and influential factors are very complicated, and urinary hydroxyl PAHs which are major metabolites of PAHs in human urine have been widely used as biomarkers for the estimation of health risks as a result of PAHs exposure. Therefore, we develop novel methods for the establishment target analysis of hydroxy metabolites and the high-throughput HPLC-FD profiling analysis of the hydroxy metabolites and their structure analogues, and build lung cancer risk prediction model of occupational exposure to PAHs, it is an important practical significance to looking for PAHs’early warning marker of lung cancer, early dagnosis, occupational safety evaluation and the in-depth study of the molecular mechanism of lung cancer.In the chapter 2, a novel method of first derivative synchronous fluorescence was developed for the rapid simultaneous analysis of trace 1-hydroxypyrene （1-OHP）, 1-naphthol （1-NAP）, 2-naphthol （2-NAP）, 9-hydroxyphenanthrene （9-OHPe） and 2-hydroxyfluorene （2-OHFlu） in human urine. In the B–R buffer solution of pH 5.0, these five metabolites of PAHs can be simultaneously identified and quantified atΔλ= 10 nm using Tween-20 as a sensitizer. The first derivative synchronous spectra were obtained by spectroscopy software program for spectral acquisition and subsequent calculation of the derivative data using the Savitzky–Golay method. The corresponding first derivative synchronous fluorescence data of 1-OHP, 1-NAP, 2-NAP, 9-OHPe and 2-OHFlu were manipulated atλ= 389.4 nm、332.4 nm、338.6 nm、355.8 nm and 306.0 nm using the peak–zero method, respectively. In the optimal experimental conditions, there was a linear relationship between the fluorescence intensity and the concentration of 1-OHP, 1-NAP, 2-NAP, 9-OHPe and 2-OHFlu in the range of 1.75×10-94.50×10-6 mol L-1, 3.64×10-82.20×10-4 mol L-1, 8.18×10-91.20×10-4 mol L-1, 3.26×10-98.50×10-5 mol L-1 and 4.88×10-9 5.50×10-6 mol L-1, respectively. The limits of detection （LOD） were found to be 5.25×10-10 mol L-1 for 1-OHP, 1.10×10-8 mol L-1 for 1-NAP, 2.46×10-9 mol L-1 for 2-NAP, 9.77×10-10 mol L-1 for 9-OHPe and 1.46×10-9 mol L-1 for 2-OHFlu. The proposed method is reliable, selective and sensitive, and has been used successfully in the determination of traces of 1-OHP, 1-NAP, 2-NAP, 9-OHPe and 2-OHFlu in human urine samples, whose results were in good agreement with those gained by the HPLC method.In the chapter 3, a method was presented for the determination of 5 polycyclic aromatic hydrocarbons （PAHs） and 6 hydroxy polycyclic aromatic hydrocarbons （OH-PAHs） by fluorescence and diode array detection - reversed phase high performance liquid chromatographic method. The determination was carried out on a Shim-pack VP - ODS C18 column （150 mm×4.6 mm, 4.6±0.3μm） by 20μL sample and Methanol/HAc- NaAc （pH 4.5） （65:35, V/V ） was used as mobile phase at a flow rate of 1.0 mL min-1 and the column oven temperature was set at 30°C. Instrument detection limits of the fluorescence detection were 0.0010.090μmol L-1 and the diode array detection were 0.0500.900μmol L-1, precisions of the method were 0.61.4% （fluorescence detection） and 1.15.5% （diode array detection）. The recoveries of standard addition were 95.5108.0%. The method is suitable for the identification and quantification of these compositions in human urine.In the chapter 4, classification models of lung cancer patients and healthy people have been built from high performance liquid chromatography data. The PAHs metabolic profiling from 36 patients with lung cancer and 10 normal adults was determined by RP-HPLC,and analyzed in the methods of principal component analysis and partial least squares, the classification model was built, finally the two more important indicator with relatively significant abnormal changes （1–NAP, NAP） were identified, it is useful for lung cancer diagnoses.