【摘要】：目的甲狀腺是人體最大的內(nèi)分泌腺,其分泌的甲狀腺激素對人體的生長發(fā)育和新陳代謝有很重要的作用。甲狀腺干擾物對人及動物的甲狀腺有很顯著的影響,能夠干擾甲狀腺功能、誘發(fā)自身免疫性甲狀腺疾病、還有潛在的致甲狀腺腫瘤的作用。在已知的甲狀腺干擾物中,農(nóng)藥占有很大的比例。殺草強作為一種非選擇性除草劑,被證實能夠干擾甲狀腺激素的合成以及導致甲狀腺增生和甲狀腺腫瘤,但是對其作用機制的研究報道很少。本研究以人甲狀腺細胞系Nthy-ori-3-1細胞為受試對象,在排除細胞毒性作用的劑量下,從基因水平探索殺草強致甲狀腺腫瘤相關(guān)的作用機制。方法實驗設(shè)計了殺草強0μg/ml、μg/ml.10pg/ml.100μg/ml四個劑量組,用含相應(yīng)受試物的培養(yǎng)基培養(yǎng)人甲狀腺細胞系Nthy-ori-3-1細胞24小時后,經(jīng)MTT法檢測細胞吸光度值,并計算細胞存活率。采用Agilent單標表達譜基因芯片對差異表達基因進行了篩選,并應(yīng)用實時定量PCR技術(shù)隨機選擇了五個差異基因驗證基因芯片結(jié)果的可靠性。結(jié)果MTT實驗結(jié)果顯示,殺草強劑量在1～100-g/ml時,Nthy-ori-3-1細胞的形態(tài)以及增殖同樣無顯著的變化,表明在此劑量下,殺草強對Nthy-ori-3-1細胞的影響與細胞毒性無關(guān)�；蛐酒Y選結(jié)果顯示,有90個基因的表達發(fā)生了顯著的變化,其中55個基因的表達上調(diào),35個基因表達下調(diào)；差異基因共影響了45個信號通路,其中大部分與腫瘤發(fā)生發(fā)展有關(guān)。實時定量PCR技術(shù)驗證差異基因表達,AGT、CA11基因顯著上調(diào)；MUC12、RAPH1、POLR1A基因顯著下調(diào),與基因芯片結(jié)果一致。結(jié)論殺草強劑量在1-100μg/ml時,其對Nthy-ori-3-1細胞的影響與細胞毒性無關(guān)。AGT、CAll、MUC12基因在甲狀腺腫瘤的發(fā)生發(fā)展中起到很重要的作用；RAPH1、POLR1A基因能夠影響細胞的內(nèi)部結(jié)構(gòu),包括蛋白質(zhì)的合成、修飾和代謝以及細胞骨架系統(tǒng)的裝配,實時定量PCR驗證結(jié)果與基因芯片結(jié)果一致。
[Abstract]:Objective thyroid gland is the largest endocrine gland in human body. Thyroid hormone secreted by thyroid gland plays an important role in growth, development and metabolism of human body. Thyroid disrupters have a significant effect on the thyroid gland of human and animals, which can interfere with thyroid function, induce autoimmune thyroid diseases, and have potential thyroid tumor effects. Pesticides account for a large proportion of known thyroid disruptions. As a non-selective herbicide, it has been proved to interfere with the synthesis of thyroid hormones and lead to thyroid hyperplasia and thyroid neoplasms. In this study, the human thyroid cell line Nthy-ori-3-1 cells were selected as subjects, and the mechanism related to the thyroid neoplasms induced by chlorpropoxide was explored at the gene level at the dose excluding cytotoxicity. Methods four dose groups of 0 渭 g / ml and 渭 g/ml.10pg/ml.100 渭 g/ml were designed. Human thyroid cell line Nthy-ori-3-1 cells were cultured on the culture medium containing the corresponding test material for 24 hours. The cell absorbance was measured by MTT method and the cell survival rate was calculated. The differentially expressed genes were screened by Agilent single standard gene microarray, and five differentially expressed genes were randomly selected by real-time quantitative PCR to verify the reliability of the results. Results the results of MTT test showed that the morphology and proliferation of Nthy-ori-3-1 cells had no significant change at the dose of 1~100-g/ml, which indicated that the effect of Xizuoqiang on Nthy-ori-3-1 cells was not related to the cytotoxicity. The results of gene chip screening showed that there were significant changes in the expression of 90 genes, of which 55 genes were up-regulated and 35 genes were down-regulated. Most of them are related to tumorigenesis and development. The differential gene expression was confirmed by real-time quantitative PCR, the AGT,CA11 gene was up-regulated and the MUC12,RAPH1,POLR1A gene was down-regulated, which was consistent with the results of gene chip. Conclusion the cytotoxicity of Nthy-ori-3-1 cells is not related to its effect on Nthy-ori-3-1 cells at a dose of 1-100 渭 g/ml. AGT,CAll,MUC12 gene plays an important role in the development of thyroid neoplasms, and RAPH1,POLR1A gene can affect the internal structure of the cells, including the synthesis of proteins. The results of modification and metabolism, assembly of cytoskeleton system, and real-time quantitative PCR were consistent with those of gene chip.
【學位授予單位】：昆明醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R114

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相關(guān)期刊論文 前1條

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本文編號：2274652