發(fā)布時間：2019-05-27 15:02

【摘要】：目的：通過制備偶氮染料毒性物質陽性暴露SD大鼠實驗動物模型，利用組織病理學切片診斷技術和免疫組織化學法觀察分析，進而探討偶氮染料對實驗動物機體的致突變損害和分子機制；采用液相色譜-串聯質譜定量分析技術，開展偶氮染料在動物體內的毒性蓄積、組織分布及毒代動力學研究，對大鼠陽性暴露的損害進行定量評估，為臨床上偶氮染料物質暴露污染提供理論基礎和實驗依據。 方法：(1)、通過制備偶氮染料陽性暴露的SD大鼠動物模型，結合病理學石蠟切片HE染色，對染料物質引起的損害進行病理學診斷分析。(2)、采用EnVision免疫組化法檢查肝臟和腎臟器官中CYP1A1基因的蛋白表達水平，從分子生物學水平討論其毒害程度和分子機制。(3)、建立了生物基質中4種染料物質的超快速液相色譜-串聯質譜（UFLC-MS/MS）的定量分析方法，并實際應用于實驗動物的組織分布、蓄積及毒代動力學的研究分析和定量評估。 結果：(1)、偶氮染料陽性處理組的大鼠肝組織均出現了不同程度的空泡癥狀，細胞間隔變厚，肝細胞脂肪變性，出現了大小不等的脂肪滴；腎臟組織出現程度不同的腎小管上皮細胞變性壞死，細胞界限模糊，對大鼠肝臟、腎臟造成了損傷危害。(2)、偶氮染料對大鼠肝臟CYP1A1表達分析結果顯示空白對照組和蘇丹染料處理組的平均陽性指數（IHC index）分別為138.89、1651.9、1220.35、490.16、1347.22，腎臟表達的陽性指數分別為17.69、295.68、128.31、196.73、400.33。說明偶氮染料處理組的肝細胞CYP1A1基因的蛋白表達水平明顯高于對照組，且具有統(tǒng)計學意義（P 0.01），表明上調了該基因的蛋白表達水平，引起毒性效應和致損傷作用。(3)、針對不同基質的生物樣品（血液、器官、組織等）成功建立了液相色譜-串聯質譜聯用檢測法（LC-MS/MS），，該方法快速、穩(wěn)定、低定量限（sub-ppb），適用于偶氮染料物質殘留定量測定的實際工作，是生物樣品中偶氮染料測定的穩(wěn)健的定量方法。(4)、在50mg/kg4種蘇丹染料的灌胃劑量下，組織分布實驗中殘留蓄積濃度值最高的是脂肪組織，含量最低的是心臟。(5)、4種偶氮染料分別于灌胃給藥0.6046、1.362、4.196、4.341h后，大鼠的血藥濃度達到最大值（Cmax分別為630.91、696.26、349.39、1304.61μg/L），在實驗大鼠體內的平均半衰期（t1/2）分別為1.208h、2.611h、3.449h、3.411h，藥-時曲線下面積值(AUC0-∞)分別為1150.03、2966.66、2706.37、10013.91μg·h/L，表明各種物質在動物機體內的生物利用度高、吸收速度快。 結論：偶氮染料陽性暴露對SD大鼠動物機體肝臟和腎臟產生了病理學損害作用，通過上調肝臟和腎臟內的CYP4501A1基因的蛋白表達水平，引起毒性效應和致損傷作用；偶氮染料在動物機體中易在組織器官內產生蓄積，脂肪組織中含量最高，毒代動力學數據表明4種染料物質的半衰期較短，生物利用度高，較易被吸收。
[Abstract]:Objective: to establish an experimental animal model of SD rats exposed to azo dye toxic substances, and to observe and analyze them by histochemical section diagnostic technique and immunohistochemistry. Furthermore, the mutagenic damage and molecular mechanism of azo dyes on experimental animals were discussed. The toxic accumulation, tissue distribution and toxicokinetics of azo dyes in animals were studied by liquid chromatography-tandem mass spectrometry (LC-MS), and the damage of positive exposure in rats was quantitatively evaluated. It provides theoretical and experimental basis for azo dye exposure to pollution in clinic. Methods: (1) the pathological diagnosis and analysis of the damage caused by dye substances were carried out by establishing the animal model of SD rats exposed to azo dye and combining with HE staining of pathological paraffin sections. (2), The protein expression of CYP1A1 gene in liver and kidney organs was detected by EnVision Immunohistochemical method, and its toxicity and molecular mechanism were discussed from the level of molecular biology. (3), A method for quantitative analysis of four dye substances in biological matrix by ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) was established and applied to the study, analysis and quantitative evaluation of tissue distribution, accumulation and toxicokinetics of experimental animals. Results: (1) in the azo dye positive group, the liver tissue of the rats showed vacuole symptoms in different degrees, the cell septum became thicker, the hepatocytes steatosis, and fat droplets of different sizes appeared. Renal tissue showed degeneration and necrosis of renal tubular epithelial cells and blurred cell boundaries, which caused damage to rat liver and kidney. (2), The results of analysis of CYP1A1 expression in rat liver by azo dye showed that the average positive index (IHC index) of blank control group and Sudan dye treatment group was 138.89, 1651.9, 1220.35490.16, 1347.22, respectively. The positive index of renal expression was 17.69295.68128.31196.73400.33. The results showed that the protein expression level of CYP1A1 gene in the group treated with azo dye was significantly higher than that in the control group (P < 0.01), indicating that the protein expression level of the gene was up-regulated. (3) liquid chromatography-tandem mass spectrometry (LC-MS/MS) was successfully established for biological samples of different matrices (blood, organs, tissues, etc.). The method is rapid and stable. Low fixed limit (sub-ppb), which is suitable for the quantitative determination of azo dye residues, is a robust quantitative method for the determination of azo dye in biological samples. (4) at the gastric perfusion dose of 50mg/kg4 Sudan dye, In the tissue distribution experiment, the residual accumulation concentration of adipose tissue was the highest, and the lowest content was the heart. (5) the four azo dyes were given 0.6046, 1.366, 4.196, 4.341 h, respectively, after intragastric administration of 0.6046, 1.362, 4.196, 4.341 h after intragastrically administration of 0.6046, 1.366, 4.196, 4.341 h, respectively. The blood concentration of rats reached the maximum (Cmax = 630.91696.26349.39, 1304.61 渭 g / L), respectively), the average half-life (T1 鈮

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