毒死蜱胁迫下斑马鱼胚胎蛋白质组的差异表达
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摘要
毒死蜱是目前世界上生产和销售量最大的有机磷杀虫剂之一,随着我国经济发展对粮食需求量的增加,粮食和经济作物的多种病害并存以及农药残留量的增加使病虫害的抗药性迅速增加,迫使农民越来越多地使用农药,同时,随着一些高毒的有机磷农药被禁止生产和使用,毒死蜱等中低毒有机磷农药被大量的使用。由毒死蜱引发的人和动物所产生的多种毒性反应已引起人们的广泛关注。
本论文选用斑马鱼这一新型的毒理模式动物,研究了毒死蜱对斑马鱼胚胎的急性毒理,在表型上观察了毒死蜱对斑马鱼胚胎发育的影响,结果显示:毒死蜱能引起很多的毒理学终点:如动物极破裂,细胞分裂不规律,外包不完全,脊柱弯曲,黑色素不全,卵黄囊病,卵凝结,尾部不分离,心率降低,孵化率降低等。同时统计了孵化率、心率、畸形等与毒死蜱浓度的相关性。为毒死蜱的合理使用提供了科学依据。
借助双向电泳、质谱分析等蛋白质组学分析方法,构建斑马鱼胚胎的蛋白质表达谱。利用Imagemaster 2D Elite 6.01分析软件,分析得到在250ppm浓度的毒死蜱胁迫下,24hpf斑马鱼胚胎产生的19个差异蛋白质,这些差异蛋白都是体积百分比变化均大于2倍甚至3倍,且符合t检验(p<0.05)的显著差异蛋白点。
通过基质辅助激光解析电离飞行时间质谱技术(MALDI-TOF-MS)获得了这些差异蛋白质的肽质量指纹图谱(PMF),并通过对斑马鱼蛋白数据库检索,初步鉴定得到4个差异蛋白,其中,下调表达蛋白三种,分别是:神经调节蛋白2b (neuregulin 2b)、肌节同源盒C(muscle segment homeobox C)、B细胞受体结合蛋白(B-cell receptor-associated protein 31);上调表达蛋白一种:酪蛋白激酶1α1-like (CSNK1A1L - casein kinase 1, alpha 1-like)。通过这些初步的质谱鉴定结果,首先我们验证了毒死蜱的传统毒理机制,即:毒死蜱对神经具有明显的毒理作用。揭示了毒死蜱能影响细胞的发育及凋亡、胞内蛋白运输、转录调控、信号转导等,使我们对毒死蜱的毒理学有了进一步的了解。在毒死蜱胁迫下,斑马鱼还能开启部分磷酸化依赖的信号通路,通过体内自我的调节以应对外来有毒物质的入侵。
综上所述,本论文对毒死蜱的毒理学在表型和分子水平上进行了综合研究,深入分析了毒死蜱的毒理学机制,揭示了毒死蜱的几种新的毒理可能靶标分子。本研究结果为进一步揭示毒死蜱的毒理代谢网络,探究毒死蜱毒理作用的机制,为减少毒死蜱对动物的毒理作用及其合理使用提供科学依据和技术支持。
Chlorpyrifos is one of the largest production and sales of organophosphorus pesticide in the world.With the economic development and increasing demands for food, the resistance of pests is increasing rapidly for increase of pesticide residues,which forces farmers to use more pesticides. Meanwhile, some high toxic organophosphate pesticides are prohibited, while chlorpyrifos and other organophosphate pesticides of low toxicity have been widely used. People began to concern human and animal toxicity caused by the chlorpyrifos.
Zebrafish ,a new model animal, was used in this paper. We studied the acute toxicity of chlorpyrifos on zebrafish embryos. Phenotype of zebrafish embryos treated by chlorpyrifos showed that: chlorpyrifos can cause a lot of toxicological endpoints, for example, animal pole broken, irregular cell division, curvature of the spine, melanin incompleted, yolk sac disease, egg condensation, no separation of the tail, heart rate decreased, and low hatchability. We also analyzed the hatching rate, heart rate, malformations associated with different chlorpyrifos concentrations. We provided a scientific basis for the rational use of chlorpyrifos.
In addition, by means of two-dimensional electrophoresis, mass spectrometry and other proteomic methods to construct zebrafish embryo protein expression profile in different periods. Under the stress of chlorpyrifos (250ppm) , we found 19 differentially expressed proteins of 24hpf zebrafish embryos. These proteins were of significant difference of protein spot and the percentage of volume change more than 2 times,sometimes up to 3 times, according to the T-test (p <0.05).
Through matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), we got these specific-expressed protein mass fingerprintings (PMFs). And through the zebrafish protein database, initially identified 4 differential-expressed proteins,3 down-regulated proteins:neuregulin 2b,muscle segment homeobox C,B-cell receptor-associated protein 31,and 1 up-regulated proteins: CSNK1A1L - casein kinase 1, alpha 1-like. We first verified the traditional toxicological mechanism of chlorpyrifos: It can cause significant toxicological effects on the nerves. We also revealed chlorpyrifos can affect cell development and apoptosis, intracellular protein transport, transcriptional regulation, signal transduction,ect. Make us have a better understanding to the toxicology of chlorpyrifos. Besides, zebrafish can also open some phosphorylation-dependent signaling pathway, through self-regulation in response to foreign toxicity invasion.
In summary, we have made a comprehensive study on the toxicology of chlorpyrifos at the phenotypic and molecular level, analysed the toxicological mechanism of chlorpyrifos and revealed several new targets for molecular toxicology, The results further uncovered the toxicity of chlorpyrifos metabolic networks, explored the mechanism of toxicity of chlorpyrifos and provided a scientific basis and technical support to reduce the toxicity of chlorpyrifos on the animal and its rational use.
本论文选用斑马鱼这一新型的毒理模式动物,研究了毒死蜱对斑马鱼胚胎的急性毒理,在表型上观察了毒死蜱对斑马鱼胚胎发育的影响,结果显示:毒死蜱能引起很多的毒理学终点:如动物极破裂,细胞分裂不规律,外包不完全,脊柱弯曲,黑色素不全,卵黄囊病,卵凝结,尾部不分离,心率降低,孵化率降低等。同时统计了孵化率、心率、畸形等与毒死蜱浓度的相关性。为毒死蜱的合理使用提供了科学依据。
借助双向电泳、质谱分析等蛋白质组学分析方法,构建斑马鱼胚胎的蛋白质表达谱。利用Imagemaster 2D Elite 6.01分析软件,分析得到在250ppm浓度的毒死蜱胁迫下,24hpf斑马鱼胚胎产生的19个差异蛋白质,这些差异蛋白都是体积百分比变化均大于2倍甚至3倍,且符合t检验(p<0.05)的显著差异蛋白点。
通过基质辅助激光解析电离飞行时间质谱技术(MALDI-TOF-MS)获得了这些差异蛋白质的肽质量指纹图谱(PMF),并通过对斑马鱼蛋白数据库检索,初步鉴定得到4个差异蛋白,其中,下调表达蛋白三种,分别是:神经调节蛋白2b (neuregulin 2b)、肌节同源盒C(muscle segment homeobox C)、B细胞受体结合蛋白(B-cell receptor-associated protein 31);上调表达蛋白一种:酪蛋白激酶1α1-like (CSNK1A1L - casein kinase 1, alpha 1-like)。通过这些初步的质谱鉴定结果,首先我们验证了毒死蜱的传统毒理机制,即:毒死蜱对神经具有明显的毒理作用。揭示了毒死蜱能影响细胞的发育及凋亡、胞内蛋白运输、转录调控、信号转导等,使我们对毒死蜱的毒理学有了进一步的了解。在毒死蜱胁迫下,斑马鱼还能开启部分磷酸化依赖的信号通路,通过体内自我的调节以应对外来有毒物质的入侵。
综上所述,本论文对毒死蜱的毒理学在表型和分子水平上进行了综合研究,深入分析了毒死蜱的毒理学机制,揭示了毒死蜱的几种新的毒理可能靶标分子。本研究结果为进一步揭示毒死蜱的毒理代谢网络,探究毒死蜱毒理作用的机制,为减少毒死蜱对动物的毒理作用及其合理使用提供科学依据和技术支持。
Chlorpyrifos is one of the largest production and sales of organophosphorus pesticide in the world.With the economic development and increasing demands for food, the resistance of pests is increasing rapidly for increase of pesticide residues,which forces farmers to use more pesticides. Meanwhile, some high toxic organophosphate pesticides are prohibited, while chlorpyrifos and other organophosphate pesticides of low toxicity have been widely used. People began to concern human and animal toxicity caused by the chlorpyrifos.
Zebrafish ,a new model animal, was used in this paper. We studied the acute toxicity of chlorpyrifos on zebrafish embryos. Phenotype of zebrafish embryos treated by chlorpyrifos showed that: chlorpyrifos can cause a lot of toxicological endpoints, for example, animal pole broken, irregular cell division, curvature of the spine, melanin incompleted, yolk sac disease, egg condensation, no separation of the tail, heart rate decreased, and low hatchability. We also analyzed the hatching rate, heart rate, malformations associated with different chlorpyrifos concentrations. We provided a scientific basis for the rational use of chlorpyrifos.
In addition, by means of two-dimensional electrophoresis, mass spectrometry and other proteomic methods to construct zebrafish embryo protein expression profile in different periods. Under the stress of chlorpyrifos (250ppm) , we found 19 differentially expressed proteins of 24hpf zebrafish embryos. These proteins were of significant difference of protein spot and the percentage of volume change more than 2 times,sometimes up to 3 times, according to the T-test (p <0.05).
Through matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), we got these specific-expressed protein mass fingerprintings (PMFs). And through the zebrafish protein database, initially identified 4 differential-expressed proteins,3 down-regulated proteins:neuregulin 2b,muscle segment homeobox C,B-cell receptor-associated protein 31,and 1 up-regulated proteins: CSNK1A1L - casein kinase 1, alpha 1-like. We first verified the traditional toxicological mechanism of chlorpyrifos: It can cause significant toxicological effects on the nerves. We also revealed chlorpyrifos can affect cell development and apoptosis, intracellular protein transport, transcriptional regulation, signal transduction,ect. Make us have a better understanding to the toxicology of chlorpyrifos. Besides, zebrafish can also open some phosphorylation-dependent signaling pathway, through self-regulation in response to foreign toxicity invasion.
In summary, we have made a comprehensive study on the toxicology of chlorpyrifos at the phenotypic and molecular level, analysed the toxicological mechanism of chlorpyrifos and revealed several new targets for molecular toxicology, The results further uncovered the toxicity of chlorpyrifos metabolic networks, explored the mechanism of toxicity of chlorpyrifos and provided a scientific basis and technical support to reduce the toxicity of chlorpyrifos on the animal and its rational use.
引文
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