本文選題：有機磷農(nóng)藥　切入點：有機氯農(nóng)藥　出處：《昆明理工大學》2016年博士論文　論文類型：學位論文

【摘要】：近年來,甲狀腺疾病的發(fā)病率有逐年上升的趨勢,環(huán)境內(nèi)分泌干擾物對甲狀腺相關(guān)疾病和人群健康的影響越來越受到廣泛關(guān)注。研究表明,甲狀腺干擾物(TCDs)可影響甲狀腺激素水平、干擾下丘腦-垂體-甲狀腺軸穩(wěn)態(tài),造成甲狀腺機能減退,甚至導致甲狀腺腫瘤的發(fā)生。作為已知甲狀腺干擾物的典型代表,農(nóng)藥類環(huán)境激素對甲狀腺內(nèi)分泌干擾效應(yīng)的流行病學調(diào)查和作用機制研究具有重要意義。本研究選取常見的幾種有機磷、有機氯農(nóng)藥為研究對象,首先針對農(nóng)藥及其代謝產(chǎn)物殘留量低、存在基質(zhì)復雜多樣的特點,建立靈敏、高效的分析方法,為研究其在環(huán)境及生物體內(nèi)的暴露水平,進行危害評估奠定基礎(chǔ)；采用建立的分析方法,結(jié)合流行病學調(diào)查的手段,從宏觀上了解特定人群有機氯農(nóng)藥體內(nèi)暴露水平對甲狀腺激素的影響；再初步從細胞分子水平研究農(nóng)藥對人甲狀腺細胞全基因表達譜的影響,以探討農(nóng)藥對甲狀腺內(nèi)分泌干擾的可能機制。主要研究內(nèi)容如下：(1)采用超聲輔助-分散液液微萃取樣品前處理技術(shù),建立了水樣中有機磷農(nóng)藥殘留檢測的氣相色譜-脈沖火焰光度檢測器法。對影響萃取效率的參數(shù)包括萃取劑和分散劑種類、體積、超聲時間、pH等進行了優(yōu)化。在最佳條件下,7種有機磷農(nóng)藥(二嗪農(nóng),乙拌磷,毒死蜱,甲基對硫磷,倍硫磷,對硫磷,喹硫磷)的檢測線性范圍為0.1-80 ng/mL,相關(guān)系數(shù)r在0.9945～0.9995之間,方法檢出限為0.01-0.04 ng/mL,定量限0.05-0.1 ng/mL,回收率為72.1%-110.0%,相對標準偏差9.5%,富集倍數(shù)為143-374。該方法具有萃取時間短,檢出限低,所需溶劑少,富集倍數(shù)高等優(yōu)點,適用于環(huán)境水樣中痕量成分的富集和分析。(2)采用超聲輔助-分散固相萃取-分散液液微萃取樣品前處理技術(shù),建立了土壤中有機磷農(nóng)藥殘留檢測的氣相色譜-脈沖火焰光度檢測器法。對分散固相萃取過程的條件以及分散液液微萃取的參數(shù)如萃取劑種類和體積、水相體積、超聲時間等進行了優(yōu)化。在最佳條件下,6種有機磷農(nóng)藥(二嗪農(nóng),乙拌磷,毒死蜱,倍硫磷,對硫磷,喹硫磷)在濃度為5～150 ng／g的范圍內(nèi)線性關(guān)系良好,相關(guān)系數(shù)r在0.9910～0.9967之間,檢出限范圍在0.2～0.5 ng／g,定量限0.5～1.2 ng／g。富集倍數(shù)為22-35倍,回收率在79.6%-106.8%之間,相對標準偏差范圍是5.3%-7.8%。該方法將分散固相萃取與分散液液微萃取技術(shù)聯(lián)用,結(jié)合兩者凈化和富集的優(yōu)勢處理復雜土壤基質(zhì)；并采用輕質(zhì)、低毒、對環(huán)境友好的溶劑異辛醇作為萃取劑,代替?zhèn)鹘y(tǒng)鹵代烴類溶劑,拓展了萃取劑種類。(3)采用甲酸沉淀蛋白,C18小柱提取、 Florisil小柱凈化分析物,建立固相萃取-氣相色譜-電子捕獲檢測器法檢測血清中有機氯農(nóng)藥殘留。對色譜條件、固相萃取柱類型、洗脫溶劑種類和體積進行優(yōu)化。在最佳條件下,正己烷+丙酮(9+1)作為提取和凈化的洗脫劑,8種有機氯農(nóng)藥(α-BHC.p-BHC、γ-BHC.δ-BHC. p,p'-DDE.p,p'-DDD.α,p'-DDT.p,p'-DDT)在濃度2～200 ng/mL范圍內(nèi)線性關(guān)系良好,相關(guān)系數(shù)r在0.9964～0.9990,檢出限為0.1～0.9 ng/mL,定量限在0.4～3.0 ng/mL,加標回收率在80.5%～112.7%之間,相對標準偏差2.1%-7.9%。該方法簡單、高效,符合農(nóng)殘柃測要求,可用于[缸清中有機氯農(nóng)藥殘留分析。(4)以昆明市呈貢區(qū)50～70歲農(nóng)村男性居民為研究對象,共選取134例樣本,采用流行病學橫斷面研究的方法,問卷調(diào)查與實驗室分析手段相結(jié)合,應(yīng)用多重線性回歸、spearman相關(guān)等統(tǒng)計方法對數(shù)據(jù)進行處理,研究血清有機氯暴露水平與甲狀腺激素水平相關(guān)性。結(jié)果表明：該人群血清有機氯農(nóng)藥殘留以p,p'-DDE為主,中位數(shù)達357.79 ng/g lw,說明該地區(qū)有機氯來源主要是歷史殘留；體質(zhì)指數(shù)BMI和年齡是影響血清有機氯含量的主要因素,并且呈正相關(guān)趨勢；相關(guān)性分析顯示,p,p'-DDE與促甲狀腺激素TSH呈正相關(guān),提示p,p'-DDE可能通過影響TSH的水平,進而影響下丘腦-垂體-甲狀腺軸的穩(wěn)態(tài)而干擾甲狀腺功能。(5)研究p,p'-DDE和馬拉硫磷對人甲狀腺細胞系Nthy-ori-3-1細胞全基因表達譜的影響。MTT實驗確定了不產(chǎn)生細胞毒性的農(nóng)藥劑量為1μg、mL,在該劑量下,馬拉硫磷組基因芯片篩選出差異表達基因為73個,其中31個基因表達上調(diào),42個基因表達下調(diào),差異基因影響的通路共為35個；p,p'-DDE組篩選出差異表達基因33個,其中20個基因表達上調(diào),13個基因表達下調(diào),差異基因影響的通路共為14個。這些差異基因主要涉及信號轉(zhuǎn)導、細胞生長和免疫調(diào)節(jié)以及細胞骨架構(gòu)建、DNA合成和轉(zhuǎn)錄、信號轉(zhuǎn)導等功能。影響細胞增殖、分化、凋亡的通路占絕大部分。與甲狀腺激素分泌合成直接相關(guān)的基因并未被發(fā)現(xiàn)。以ACTB為內(nèi)參基因,實時定量PCR隨機驗證馬拉硫磷組11個、p,p'-DDE組4個差異基因表達,最終結(jié)果均與基因芯片結(jié)果一致。
[Abstract]:In recent years, the incidence of thyroid disease has increased year after year, environmental endocrine disruptors on thyroid related diseases and human health effect has attracted more and more attention. The research results show that thyroid disruptors (TCDs) can affect the level of thyroid hormone, disturbance of hypothalamic pituitary thyroid axis homeostasis caused by hypothyroidism, even lead to thyroid the occurrence of tumor. As a typical representative of known thyroid disruptors, has important significance in epidemiological investigation and study the mechanism of pesticide environmental hormone effects on thyroid endocrine disturbance. This study selected several common organophosphorus, organochlorine pesticides as the research object, first for the pesticide and its metabolites residues are low, characteristics of complex matrix the establishment of sensitive and efficient analysis, to investigate the environment and organism level of exposure and risk of And lay the foundation for the assessment; the establishment of analytical methods, combined with epidemiological investigation methods, to understand the effect of organochlorine pesticide exposure level of specific groups of thyroid hormone from the macro; and preliminary from cellular and molecular level of spectrum of the effects of pesticides on the expression of human thyroid cell gene, to explore the possible mechanism of pesticide endocrine disturbance the main contents are as follows: (1) using ultrasound assisted dispersive liquid-liquid microextraction sample pretreatment technique, was developed for the detection of the residues of organophosphorus pesticides in water gas chromatography pulsed flame detector. The method of parameters affecting the extraction efficiency including the extraction agent and dispersing agent, volume, ultrasonic time, pH were optimized. Under the optimal conditions, 7 kinds of organophosphorus pesticides (two diazinon, disulfoton, chlorpyrifos, methyl parathion, parathion, fenthion, quetiapine P) linear detection The range of 0.1-80 ng/mL, the correlation coefficient r is 0.9945 ~ 0.9995, the detection limit is 0.01-0.04 ng/mL 0.05-0.1 ng/mL, the limit of quantification, the recovery rate was 72.1%-110.0%, the relative standard deviation of 9.5% 143-374., enrichment factor is the method has short extraction time, low detection limit, required less solvent, high enrichment factor, enrichment and analysis suitable for trace elements. (2) using ultrasound assisted dispersive solid-phase extraction and micro - Extraction Pretreatment of dispersion liquid, established the detection of residues of organophosphorus pesticides in soil gas chromatography pulsed flame photometric detector. The dispersive solid phase extraction process conditions and dispersive liquid-liquid microextraction the parameters such as extraction solvent type and volume, water volume, ultrasonic time were optimized. Under the optimal conditions, 6 kinds of organophosphorus pesticides (two diazinon, disulfoton, chlorpyrifos, parathion, sulfur and phosphorus times, quintiofos) At the concentration of 5~150 ng / g range a good linear relationship, correlation coefficient r is 0.9910 ~ 0.9967, the detection limit in the range of 0.2 ~ 0.5 ng / g, the quantitative limit of 0.5 ~ 1.2 ng / g. concentration ratio of 22-35 times, the recovery rate was 79.6%-106.8%, the relative standard deviation is in the range of 5.3% -7.8%. in the method dispersive solid phase extraction and dispersive liquid-liquid microextraction coupled with both purified and enriched the advantages of dealing with complex soil matrix; and the use of lightweight, low toxic, environmentally friendly solvent of isooctyl alcohol as extractant, instead of halogenated hydrocarbon solvent, development of extractants. (3) the acid precipitated protein, C18 column extraction, analyte purification Florisil column, a solid phase extraction and gas chromatography with electron capture detector to detect serum organochlorine pesticide residues method. The chromatographic conditions, solid phase extraction column type, the optimal solvent type and volume of elution . under the optimum conditions, hexane and acetone (9+1) as the extraction and purification of eluent, 8 kinds of organochlorine pesticides (alpha gamma delta -BHC. -BHC.p-BHC, -BHC. P, p'-DDE.p, p'-DDD. alpha, p'-DDT.p, p'-DDT) in the concentration range of 2~200 ng/mL good linear relationship, the correlation coefficient r between 0.9964 and 0.9990. The detection limit is 0.1 ~ 0.9 ng/mL, the limit of quantitation in 0.4 ~ 3 ng/mL, the recovery rate is from 80.5% to 112.7% between the standard, the relative standard deviation of the 2.1%-7.9%. method is simple, efficient, meets the measurement requirements for pesticide residues Eurya, analysis of organochlorine pesticide residues in the bathtub. "(4) in Kunming District of Chenggong city at the age of 50~70 rural male residents as the research object, a total of 134 cases of samples, cross-sectional study using epidemiological methods, questionnaire survey and laboratory analysis methods combined, using multiple linear regression, Spearman correlation statistical methods for data processing, the study of serum organochlorine exposure dew Flat the level of thyroid hormone and correlation. The results showed that the serum organochlorine pesticide residues in P, p'-DDE, median reached 357.79 ng/g LW, to indicate the source of organic chlorine in the region is mainly historical residue; body mass index BMI and age are the main factors affecting the organic chlorine content in serum, and the tendency of positive correlation; correlation analysis showed P, p'-DDE, correlation, and promoting positive thyroid hormone TSH suggested that P, p'-DDE may influence the level of TSH, thereby affecting the hypothalamic pituitary thyroid axis and the steady state interference. The thyroid function (5) of P, p'-DDE and malathion on effects of.MTT of the experimental spectra confirmed that the pesticide dose does not produce cytotoxicity was 1 g, Nthy-ori-3-1 cell gene expression in human thyroid cell line mL in the dose, malathion group gene microarray selected153differentially expressed 73 genes, including 31 up-regulated genes and 42 A gene expression pathway, differential gene effects for a total of 35; P, p'-DDE group identified 33 differentially expressed genes, including 20 up-regulated genes and 13 downregulated genes, gene pathway effects for a total of 14. These differentially expressed genes were mainly involved in signal transduction, cell growth and immune regulation and cell skeleton construction, DNA synthesis and transcription function, signal transduction etc. affect cell proliferation, differentiation, apoptosis pathway andcontainermetaphoraccountforthemajorpartofthetotalmetaphorsinthedata. Thyroid hormone secretion and directly related to the synthesis of the gene has not been found. Using ACTB as the reference gene, real time quantitative PCR random verification malathion group 11, P group, p'-DDE gene expression in 4 a difference in the end result results with the gene chip.

【學位授予單位】：昆明理工大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R581;X592

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本文編號：1558385