纳米银引起斑马鱼胚胎色素减退的分子机制
发布时间：2019-03-28 15:38
【摘要】：纳米银有着广谱的抑菌特性,被广泛的应用于人类的生产和生活中,而纳米银生物安全性评估的工作还远远不够。最近有研究发现纳米银能够抑制脊椎动物红系细胞的形成和分化,引起脊柱弯曲和心率失常等现象。但是在模式生物中研究纳米银影响神经脊发育和色素细胞生成的不多。在本实验中,选取模式生物斑马鱼(Danio rerio)作为研究的对象。首先通过基因芯片技术和生物信息学手段筛选出纳米银暴露斑马鱼胚胎中和色素形成相关的表达下调的基因,接着利用qRT-PCR技术和整体原位杂交技术验证基因表达下调的结果。然后利用黑色素生成激活剂棕榈酸和双丁酰环磷腺苷来营救(恢复)纳米银暴露组胚胎色素形成相关基因的表达下调,探究色素减退的分子机制。最后,利用银螯合剂L-半胱氨酸来区分纳米银生物学效应的来源。主要结果如下:1.纳米银引起斑马鱼胚胎色素减退纳米银组胚胎相对于上清液组胚胎和野生型组胚胎出现了色素减退的现象,黑色素细胞显著的减少和黄色素细胞略微的减少。2.纳米银抑制黑色素细胞和黄色素细胞相关基因的转录通过基因芯片技术和生物信息学手段筛选出的16个和斑马鱼色素形成相关的基因呈现下调表达。进一步通过qRT-PCR技术检测了纳米银暴露组胚胎中黑色素细胞形成相关基因mitfa、tyrp1b、oca2、dct,黄色素细胞形成相关基因gch2、aox5,银色素细胞形成相关基因csf1b的表达量。和对照组相比,纳米银组和银离子组胚胎中基因mitfa、tyrp1b、oca2、dct、gch2和aox5均出现显著性的表达下调,但是基因csf1b却出现了显著性的上调表达。此外,本研究利用整体原位杂交技术检测了纳米银暴露组胚胎中上述相关基因的转录水平和时空分布。和野生型组胚胎相比,纳米银暴露组胚胎和银离子组胚胎中黑色素细胞相关基因dct和tyrp1b均出现显著性的表达下调。3.纳米银主要通过释放银离子影响神经脊的形成和色素细胞的形成本实验首先检测了不同浓度的棕榈酸和双丁酰环腺苷酸对胚胎色素细胞生成相关基因表达量的影响。最终选取了10μM双丁酰环腺苷酸和1μM棕榈酸进行接下来的联合暴露实验。纳米银暴露组和银离子组胚胎中基因crestin、mitfa、dct和gch2的表达均受到显著性的抑制,但是在添加了双丁酰环腺苷酸或棕榈酸中后上述基因恢复到正常水平甚至更高的水平。L-半胱氨酸能够鳌合银,本研究发现纳米银组胚胎中出现了黑色素细胞和黄色素细胞的减少,然而,在纳米银和L-半胱氨酸联合暴露组胚胎中黄色素细胞和黑色素细胞又开始增多。通过整体原位杂交技术检测发现纳米银和L-半胱氨酸联合暴露组胚胎相比较纳米银暴露组胚胎,基因dct和gch2的表达量恢复增多。根据以上实验结果推测纳米银主要通过释放银离子来抑制神经脊的形成从而抑制色素细胞的生成,特异的抑制神经脊起源的黑色素细胞和黄色素细胞的形成和分化。此外,纳米银很可能在基因级联反应中通过和基因mitfa和pax7的上游因子作用来分别调控黑色素细胞和黄色素细胞的形成和分化。
[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.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X503.225
本文编号：2449005
[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.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X503.225
【参考文献】
相关期刊论文 前1条
1 邹苏琪;殷梧;杨昱鹏;陈林;胡兵;;斑马鱼行为学实验在神经科学中的应用[J];生物化学与生物物理进展;2009年01期
，本文编号：2449005
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