【摘要】：納米銀有著廣譜的抑菌特性,被廣泛的應(yīng)用于人類(lèi)的生產(chǎn)和生活中,而納米銀生物安全性評(píng)估的工作還遠(yuǎn)遠(yuǎn)不夠。最近有研究發(fā)現(xiàn)納米銀能夠抑制脊椎動(dòng)物紅系細(xì)胞的形成和分化,引起脊柱彎曲和心率失常等現(xiàn)象。但是在模式生物中研究納米銀影響神經(jīng)脊發(fā)育和色素細(xì)胞生成的不多。在本實(shí)驗(yàn)中,選取模式生物斑馬魚(yú)(Danio rerio)作為研究的對(duì)象。首先通過(guò)基因芯片技術(shù)和生物信息學(xué)手段篩選出納米銀暴露斑馬魚(yú)胚胎中和色素形成相關(guān)的表達(dá)下調(diào)的基因,接著利用qRT-PCR技術(shù)和整體原位雜交技術(shù)驗(yàn)證基因表達(dá)下調(diào)的結(jié)果。然后利用黑色素生成激活劑棕櫚酸和雙丁酰環(huán)磷腺苷來(lái)營(yíng)救(恢復(fù))納米銀暴露組胚胎色素形成相關(guān)基因的表達(dá)下調(diào),探究色素減退的分子機(jī)制。最后,利用銀螯合劑L-半胱氨酸來(lái)區(qū)分納米銀生物學(xué)效應(yīng)的來(lái)源。主要結(jié)果如下:1.納米銀引起斑馬魚(yú)胚胎色素減退納米銀組胚胎相對(duì)于上清液組胚胎和野生型組胚胎出現(xiàn)了色素減退的現(xiàn)象,黑色素細(xì)胞顯著的減少和黃色素細(xì)胞略微的減少。2.納米銀抑制黑色素細(xì)胞和黃色素細(xì)胞相關(guān)基因的轉(zhuǎn)錄通過(guò)基因芯片技術(shù)和生物信息學(xué)手段篩選出的16個(gè)和斑馬魚(yú)色素形成相關(guān)的基因呈現(xiàn)下調(diào)表達(dá)。進(jìn)一步通過(guò)qRT-PCR技術(shù)檢測(cè)了納米銀暴露組胚胎中黑色素細(xì)胞形成相關(guān)基因mitfa、tyrp1b、oca2、dct,黃色素細(xì)胞形成相關(guān)基因gch2、aox5,銀色素細(xì)胞形成相關(guān)基因csf1b的表達(dá)量。和對(duì)照組相比,納米銀組和銀離子組胚胎中基因mitfa、tyrp1b、oca2、dct、gch2和aox5均出現(xiàn)顯著性的表達(dá)下調(diào),但是基因csf1b卻出現(xiàn)了顯著性的上調(diào)表達(dá)。此外,本研究利用整體原位雜交技術(shù)檢測(cè)了納米銀暴露組胚胎中上述相關(guān)基因的轉(zhuǎn)錄水平和時(shí)空分布。和野生型組胚胎相比,納米銀暴露組胚胎和銀離子組胚胎中黑色素細(xì)胞相關(guān)基因dct和tyrp1b均出現(xiàn)顯著性的表達(dá)下調(diào)。3.納米銀主要通過(guò)釋放銀離子影響神經(jīng)脊的形成和色素細(xì)胞的形成本實(shí)驗(yàn)首先檢測(cè)了不同濃度的棕櫚酸和雙丁酰環(huán)腺苷酸對(duì)胚胎色素細(xì)胞生成相關(guān)基因表達(dá)量的影響。最終選取了10μM雙丁酰環(huán)腺苷酸和1μM棕櫚酸進(jìn)行接下來(lái)的聯(lián)合暴露實(shí)驗(yàn)。納米銀暴露組和銀離子組胚胎中基因crestin、mitfa、dct和gch2的表達(dá)均受到顯著性的抑制,但是在添加了雙丁酰環(huán)腺苷酸或棕櫚酸中后上述基因恢復(fù)到正常水平甚至更高的水平。L-半胱氨酸能夠鰲合銀,本研究發(fā)現(xiàn)納米銀組胚胎中出現(xiàn)了黑色素細(xì)胞和黃色素細(xì)胞的減少,然而,在納米銀和L-半胱氨酸聯(lián)合暴露組胚胎中黃色素細(xì)胞和黑色素細(xì)胞又開(kāi)始增多。通過(guò)整體原位雜交技術(shù)檢測(cè)發(fā)現(xiàn)納米銀和L-半胱氨酸聯(lián)合暴露組胚胎相比較納米銀暴露組胚胎,基因dct和gch2的表達(dá)量恢復(fù)增多。根據(jù)以上實(shí)驗(yàn)結(jié)果推測(cè)納米銀主要通過(guò)釋放銀離子來(lái)抑制神經(jīng)脊的形成從而抑制色素細(xì)胞的生成,特異的抑制神經(jīng)脊起源的黑色素細(xì)胞和黃色素細(xì)胞的形成和分化。此外,納米銀很可能在基因級(jí)聯(lián)反應(yīng)中通過(guò)和基因mitfa和pax7的上游因子作用來(lái)分別調(diào)控黑色素細(xì)胞和黃色素細(xì)胞的形成和分化。
[Abstract]:The nano-silver has a broad-spectrum antibacterial property, is widely used in the production and the life of human beings, and the work of the nano-silver biological safety assessment is not enough. Recently, it has been found that the nano-silver can inhibit the formation and differentiation of the erythroid cells in the vertebrate and cause the phenomena of spinal curvature and arrhythmia. However, the study on the effect of nano-silver on the development of the neural ridge and the generation of pigment cells in the model organism. In this experiment, the model bizebrafish (Danio rino) was selected as the subject of the study. The expression down-regulated genes of nano-silver exposed zebrafish embryos and the pigment formation are screened through the gene chip technology and the bioinformatics method, and then the results of the down-regulation of the gene expression are verified by using the qRT-PCR technique and the whole in-situ hybridization technique. Then, the expression of the related genes of the embryonic pigment formation related genes of the nano-silver exposed group is reduced and the molecular mechanism of the hypopigmentation is explored by using the melanin generation activator palmitic acid and the dibutyrosine cyclophosphine to rescue (restore) the expression of the related genes of the nano-silver exposed group of the embryonic pigment. In the end, the origin of the biological effect of the nano-silver is distinguished by using the L-cysteine of the silver-condensing agent. The main results are as follows:1. The effect of nano-silver on the depigmentation of the embryonic and wild-type group of the Zebrafish embryo and the embryo of the wild-type group caused by nano-silver, the remarkable decrease of the melanocytes and the slight decrease of the yellow pigment. The transcription of the related genes of the melanocytes and the yellow pigment cell related genes of the nano-silver inhibited the expression of the genes related to the formation of the 16 and zebrafish pigment formed by the gene chip technology and the bioinformatics means. In that invention, the related gene mitfa, tyrp1b, oc2, dct, yellow pigment, and the related gene gch2, aox5 and the silver pigment cell of the nano-silver exposed group embryo are further detected by the qRT-PCR technique to form the expression amount of the related gene csf1b. Compared with the control group, the expression of mitfa, tyrp1b, oc2, dct, gch2 and aox5 in the nano-silver group and the silver-ion group was down-regulated. In addition, the transcription level and space-time distribution of the above-mentioned related genes in the embryo of the nano-silver exposed group were detected by the whole in situ hybridization technique. Compared with the wild-type group, there was a significant decrease in the expression of the related gene dct and tyrp1b of the melanocyte-related genes in the embryonic and silver-ion group embryos of the nano-silver exposed group. In this experiment, the effect of different concentrations of palmitic acid and dibutylenic acid on the production of the related gene was first detected by releasing the silver ions to the formation of the neural ridge and the formation of the pigment cells. The next joint exposure experiment was carried out with 10.mu. M of dicine and 1. m u.M of palmitic acid. The expression of crestin, mitfa, dct, and gch2 in both the nano-silver exposed group and the silver-ion group was significantly inhibited, but the above-mentioned genes were recovered to a normal level or even higher after the addition of the double-eugenol or palmitic acid. L-cysteine can be combined with silver. In this study, the decrease of melanocytes and yellow pigment cells in the embryo of the nano-silver group was found, however, the yellow pigment cells and the melanocytes in the embryo of the combination of nano-silver and L-cysteine also started to increase. The results showed that the expression of gene dct and gch2 increased in the exposed group of nano-silver and L-cysteine. According to the above experimental results, it is suggested that the nano-silver is mainly used for inhibiting the formation of the nerve ridge by releasing the silver ions so as to inhibit the generation of the pigment cells, and specifically inhibit the formation and differentiation of the melanocytes and the yellow pigment cells from the origin of the nerve ridge. In addition, the nano-silver is likely to control the formation and differentiation of the melanocytes and the yellow pigment cells by the action of the upstream factors of the gene mitfa and the pax7 in the gene cascade reaction.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：X503.225

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相關(guān)期刊論文 前1條

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本文編號(hào)：2449005