毒死蜱对斑马鱼胚胎的毒性机制研究

发布时间：2018-06-06 01:19

本文选题：毒死蜱 + 斑马鱼胚胎　；参考：《中国农业科学院》2015年硕士论文

【摘要】：毒死蜱是一种典型的有机磷杀虫剂,广泛应用于农业生产。随着污染事件的增多,毒死蜱复杂的生物学效应逐渐引起大家的关注。本研究主要以斑马鱼胚胎和幼鱼作为研究对象,从表型、分子水平尤其是组学水平上探究毒死蜱的环境生物学效应及其作用机理。以下是主要的研究方法和结果:1.配制高、低两种浓度梯度的毒死蜱进行急性毒性实验,其中低浓度梯度为0、0.10、0.25、0.50、0.75和1.00 ppm;高浓度梯度为0、1.00、2.00、3.00和4.00 ppm,每个浓度设置3个平行组。结果显示,处理48 h后低浓度梯度处理组胚胎孵化快于对照组,并且胚胎的孵化率与处理浓度成正相关,且未出现明显畸形。处理60 h后,各组胚胎几乎全部孵化,而且胚胎存活状况良好;在高浓度毒死蜱梯度处理组中,胚胎在48 h已经基本全部孵化,并且出现了体轴弯曲、心包囊肿等畸形现象,并且从处理开始陆续出现胚胎死亡,60 h时2 ppm处理组胚胎接近半致死。2.为了探究低浓度毒死蜱的内分泌干扰效应,使用流式细胞仪分析了其对人子宫内膜癌细胞HEC-1B生长周期的影响。结果发现毒死蜱对HEC-1B具有促增殖作用,并且同等浓度下其作用不如雌二醇,具有类似雌激素的效应。毒死蜱能够明显影响斑马鱼胚胎雌激素相关基因VTG和ERα、细胞增殖相关基因c-myc、cyclin D1、c-fos和c-jun的表达水平。吖啶橙染色结果表明0.75和1.00 ppm毒死蜱处理组中,可以观察到凋亡细胞,而细胞凋亡相关基因Bcl-2和Bax的表达水平出现紊乱。考虑到毒死蜱的神经毒性,本研究从基因的转录水平探究其神经发育毒性。3.本研究采用转录组学和蛋白质组学对其进行生物信息学分析。其中,转录组学采用RNA-seq技术,蛋白质组学采用iTRAQ技术。使用非致死低浓度毒死蜱(0.05 ppm)连续处理斑马鱼胚胎9天后进行组学分析。转录组数据分析差异基因和通路,蛋白质组数据分析差异蛋白和通路,并且进行转录组和蛋白质组联合分析。本研究结果表明:低浓度的毒死蜱具有内分泌干扰效应,能提高癌细胞的增殖系数。其加速斑马鱼胚胎孵化脱膜的原因,可能是促进细胞的生长和分裂,抑制细胞的凋亡。而高浓度的毒死蜱对斑马鱼主要表现为神经毒性,能够造成胚胎的畸形和死亡,并且显著影响神经系统相关基因的表达量。毒死蜱导致斑马鱼幼鱼的代谢、转录的相关基因和蛋白表达上调,从而影响其发育进程;导致免疫系统、环境适应性相关基因和蛋白表达下调,可能造成免疫力降低,最终导致斑马鱼幼鱼死亡。另外,毒死蜱处理产生差异表达异常显著的基因和蛋白可以作为潜在生物标记进行下一步研究。
[Abstract]:Chlorpyrifos is a typical organophosphorus insecticide, widely used in agricultural production. With the increase of pollution events, the complex biological effects of chlorpyrifos have attracted more and more attention. In this study, the environmental biological effects of chlorpyrifos and its mechanism were studied from phenotypic and molecular level, especially at the group level, with zebrafish embryos and young fish as the research objects. Here are the main research methods and results: 1. The acute toxicity test of chlorpyrifos with high and low concentration gradient was carried out. The low concentration gradient was 0.100.250.50,0.50,0.75 and 1.00 ppm.The high concentration gradient was 00.000.002.003.00 and 4.00 ppms. each concentration was divided into 3 parallel groups. The results showed that the embryo hatched faster in the low concentration gradient treatment group than that in the control group after 48 h treatment, and the hatching rate of the embryos was positively correlated with the treatment concentration, and there was no obvious abnormality. After 60 h treatment, almost all the embryos hatched, and the embryo survived well. In the high concentration chlorpyrifos gradient treatment group, the embryos had almost all hatched at 48 h, and some abnormal phenomena such as body axis bending and pericardial cyst appeared. And the embryo death occurred at 60 h after treatment. The embryo of 2 ppm treatment group was close to half lethal. 2. In order to investigate the endocrine disrupting effect of chlorpyrifos at low concentration, the effects of chlorpyrifos on the HEC-1B growth cycle of human endometrial cancer cells were analyzed by flow cytometry. The results showed that chlorpyrifos could promote the proliferation of HEC-1B, and at the same concentration, the effect of chlorpyrifos was inferior to that of estradiol, which was similar to that of estrogen. Chlorpyrifos could significantly affect the expression levels of estrogen related genes VTG and ER 伪, cell proliferation related genes c-myc cyclin D1 c-fos and c-jun in zebrafish embryos. The results of acridine orange staining showed that apoptotic cells could be observed in chlorpyrifos treated with 0. 75 and 1. 00 ppm, while the expression of apoptosis-related genes Bcl-2 and Bax were disturbed. Considering the neurotoxicity of chlorpyrifos, we investigated the neurodevelopmental toxicity of chlorpyrifos from the transcriptional level. In this study, transcriptome and proteomics were used for bioinformatics analysis. Among them, RNA-seq technique was used in transcriptome and iTRAQ technique was used in proteomics. The zebrafish embryos were treated with non-lethal low concentration chlorpyrifos (0.05 ppm) for 9 days. Transcriptional data were used to analyze differential genes and pathways, proteome data were used to analyze differential proteins and pathways, and transcriptome and proteome were combined. The results showed that low concentration of chlorpyrifos had endocrine disrupting effect and could improve the proliferative coefficient of cancer cells. The reason of accelerating the hatching and demembering of zebrafish embryos may be to promote cell growth and division and inhibit cell apoptosis. The high concentration of chlorpyrifos mainly showed neurotoxicity to zebrafish, which caused embryo deformity and death, and significantly affected the expression of genes related to the nervous system. Chlorpyrifos lead to the metabolism of juvenile zebrafish and up-regulate the expression of transcription related genes and proteins, which affect the development of zebrafish, and lead to the down-regulation of the immune system and the expression of environment-adaptive genes and proteins, which may lead to the decrease of immunity. The result is the death of juvenile zebrafish. In addition, Chlorpyrifos treatment produces abnormal differentially expressed genes and proteins that can be used as potential biomarkers for further research.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X174

【共引文献】