模式生物秀丽隐杆线虫评价真菌毒素毒性毒理机制的研究
发布时间：2018-08-31 08:30
【摘要】：真菌毒素是一类由真菌产生的有毒次级代谢产物,通过污染食品进入人类和动物体内引发癌症、不孕等症状,有严重毒性,不仅造成经济损失还同时危害人类健康。近年来对于真菌毒素毒性的研究报道较多但由于模式生物个体之间生物可及性和生物利用率之间存在差异,造成真菌毒素在不同模式生物之间毒性效应及毒理相关机制报道常有冲突。与其他模式生物比较模式生物秀丽隐杆线虫身体构造简单、遗传模式保守可避免个体差异有利于毒性评价的标准化。目前秀丽隐杆线虫已经成为评价外源性毒素毒性以及毒理机制的首选。因此本研究以秀丽隐杆线虫为模式试验动物,建立真菌毒素特别是玉米赤霉烯酮毒性评价模型,观察真菌毒素对秀丽线虫的毒性效应,选择合适评价指标并比较玉米赤霉烯酮对秀丽线虫基因表达谱的影响,深入探讨玉米赤霉烯酮对秀丽线虫在分子水平的毒性作用机制。具体研究内容及结果如下:1.采用急性中毒死亡率、身体长度、后代数目和最长寿命为评价指标建立秀丽隐杆线虫毒性评价模型,探究黄曲霉毒素B1、脱氧雪腐镰刀菌烯醇、伏马菌素、T-2毒素和玉米赤霉烯酮对秀丽线虫的毒性效应。结果显示所用的五种真菌毒素对线虫发育、生殖能力有显著抑制作用并缩短线虫寿命,可显著降低线虫成虫的体长、体宽和后代数目。T-2毒素对于急性中毒死亡率指标最为敏感,半数效应剂量1.38 mg/L。黄曲霉毒素B1是对生殖指标最敏感的的毒素,其次是伏马菌素和玉米赤霉烯酮。综合比较寿命指标是线虫对真菌毒素最为敏感的指标。2.对玉米赤霉烯酮染毒线虫使用DIC显微镜观察,结果显示染毒组线虫性腺臂发生明显萎缩,卵母细胞数目和大小均受玉米赤霉烯酮显著抑制影响,线虫世代时间显著延长。且可见染毒组内部分线虫产卵器发育畸形,畸形比率与玉米赤霉烯酮存在剂量效应关系,进一步表明玉米赤霉烯酮存在严重生殖毒性效应。3.在热胁迫刺激下,玉米赤霉烯酮可显著降低秀丽线虫对热刺激的抗性,降低受热后线虫存活几率。并且染毒线虫行为能力明显受到玉米赤霉烯酮影响,暴露48 h以上线虫头部摆动和身体弯曲频率显著降低。结果表明玉米赤霉烯酮可导致秀丽线虫对外界刺激抗性减弱、行为能力产生严重缺陷,具有神经毒性效应。4.对秀丽线虫采用亲代染毒,后代断毒恢复实验,观察实验结果显示,玉米赤霉烯酮暴露导致的发育缺陷和生殖缺陷在线虫世代间具有可传递性。在后代线虫中,发育缺陷仅得到有限的恢复,而生殖缺陷没有得到明显恢复。另外连续对三代线虫暴露玉米赤霉烯酮,毒性效应不是连续的增加或减弱,而是第二代线虫产生最为严重的毒性效应,说明线虫对玉米赤霉烯酮可产生一定代偿作用。这些结果表明玉米赤霉烯酮具有一定致畸毒性效应。5.为进一步阐明玉米赤霉烯酮对秀丽线虫毒性的相应调控机制,采用Microarray技术分析比较线虫基因表达的差异。结果显示10、40和80 mg/L剂量玉米赤霉烯酮染毒后秀丽线虫异常显著差异表达基因数目分别为171、245、3149个(与对照组相差2倍及以上),这些基因通过GO分析功能集中在磷酸化细胞信号传导、物质代谢、胚胎发育、神经细胞增殖分化、寿命调控和行为调控。统计分析受到显著影响的信号通路有表皮胶原蛋白合成信号通路、Wnt-β-Catenin信号通路和DAF-2胰岛素信号通路。基因分析结果显示玉米赤霉烯酮具有发育毒性、生殖毒性、细胞毒性和神经毒性。6.Microarray和RT-PCR结果显示表皮胶原蛋白合成信号通路的dpy-17、col-121、hch-1明显受到玉米赤霉烯酮抑制下调,同时dpy-31和sqt-3基因被刺激上调。玉米赤霉烯酮染毒组DPY-31#GFP标记线虫的荧光表达量显著高于正常线虫,在蛋白水平表明ZEN上调DPY-31表达量。同时突变体线虫对体长和后代数目评价指标比野生型线虫也更加敏感。这些结果显示玉米赤霉烯酮抑制秀丽线虫表皮胶原蛋白合成信号通路表达。7.Microarray结果和RT-PCR结果基本一致,显示10个DAF-2 insulin-like信号通路的相关基因和5个通路下游基因受到玉米赤霉烯酮染毒影响,其中daf-2、age-1、akt-1、akt-2、sgk-1、pdk-1、daf-16、dct-15、daf-21和ins-11基因上调,daf-18、hsp-1、hsf-1、dao-5和egl-4基因下调。DAF-16#GFP在线虫各个组织细胞的细胞核和细胞质中均有表达。在热刺激条件下,正常组线虫中的DAF-16#GFP迅速完成从细胞质到细胞核的转移但是玉米赤霉烯酮染毒组线虫的DAF-16#GFP细胞核富集明显受阻。并且在热刺激后的恢复过程中,染毒组线虫的DAF-16#GFP从细胞核的释放过程也明显受阻。这些结果显示玉米赤霉烯酮诱导秀丽线虫DAF-2 insulin-like信号通路表达上调。综上所述,秀丽隐杆线虫多个评价指标对真菌毒素毒性作用敏感,是优秀的评价真菌毒素毒性及其毒理机制的模式生物。同时本研究结果显示玉米赤霉烯酮对于线虫的生殖、发育毒性以及神经毒性很可能是通过影响表皮胶原蛋白合成通路和DAF-2insulin-like信号通路的正常表达实现,为进一步探究玉米赤霉烯酮毒性调节机制提供了科学依据。
[Abstract]:Mycotoxin is a kind of toxic secondary metabolites produced by fungi. It can cause cancer, infertility and other symptoms by contaminating food into humans and animals. It not only causes economic losses but also endangers human health. In recent years, there are many reports on mycotoxin toxicity, but also the organisms among model organisms. There are differences in accessibility and bioavailability, resulting in conflicting reports on toxic effects and toxicological mechanisms of mycotoxins among different model organisms. Compared with other model organisms, C. elegans is a simple model organism with a conservative genetic model that avoids individual differences and is conducive to standardization of toxicity assessment. Caenorhabditis elegans has become the first choice to evaluate the toxicity and toxicological mechanism of exogenous toxins. Therefore, the toxicity evaluation model of mycotoxins, especially zearalenone, was established to observe the toxic effect of mycotoxins on Caenorhabditis elegans, select the appropriate evaluation index and compare the corn red. The effects of mycophenone on gene expression profiles of Caenorhabditis elegans were investigated to explore the molecular toxicity mechanism of zearalenone on Caenorhabditis elegans. Specific research contents and results were as follows: 1. Toxicity evaluation model of Caenorhabditis elegans was established by using acute poisoning mortality, body length, offspring number and longevity as evaluation indicators. Toxic effects of aflatoxin B1, deoxynivalenol, fumonisin, T-2 toxin and zearalenone on Caenorhabditis elegans were studied. The results showed that the five mycotoxins had significant inhibitory effects on nematode development, reproductive capacity and longevity, and significantly reduced the length, width and number of offspring of adult nematodes. Aflatoxin B1 was the most sensitive toxin to reproductive indicators, followed by fumonisin and zearalenone. Comprehensive life span index was the most sensitive indicator to mycotoxins. 2. DIC microscope was used for zearalenone-infected nematodes. The results showed that the gonadal arms of nematodes in the exposed group atrophied significantly, the number and size of oocytes were significantly inhibited by zearalenone, and the generation time of nematodes was significantly prolonged. Zearalenone significantly reduced the resistance of C. elegans to heat stimulation and the survival rate of C. elegans after exposure to heat. Moreover, the behavioral ability of C. elegans was significantly affected by zearalenone, and the frequency of head swing and body bending significantly decreased after exposure for more than 48 hours. The results showed that zearalenone could weaken the resistance of C. elegans to external stimuli and cause serious behavioral defects, which had neurotoxic effects. 4. The growth and reproductive defects of C. elegans were observed in the adult worms after parental exposure to zearalenone. In the next generation of nematodes, only a limited recovery of developmental defects and no significant recovery of reproductive defects were observed. In addition, the toxic effect of the third generation of nematodes exposed to zearalenone was not continuous increase or decrease, but the most serious toxic effect of the second generation of nematodes, indicating that nematodes had the most serious toxic effect on zearalenone. These results indicate that zearalenone has teratogenic toxicity. 5. To further elucidate the mechanism of zearalenone's toxicity to Caenorhabditis elegans, microarray technique was used to analyze and compare the differences in gene expression of nematodes. There were 171,245,3149 abnormally differentially expressed genes in C. elegans after poisoning (2 times and more than that in the control group). These genes were mainly involved in signal transduction of phosphorylated cells, material metabolism, embryonic development, proliferation and differentiation of nerve cells, regulation of life span and behavior regulation by GO analysis. Gene analysis showed that zearalenone had developmental toxicity, reproductive toxicity, cytotoxicity and neurotoxicity. 6. Microarray and RT-PCR results showed that dpy-17, col-121, hch-1 were the epithelial collagen synthesis signaling pathways. The fluorescence expression of DPY-31 # GFP-labeled nematodes was significantly higher than that of normal nematodes, and the expression of DPY-31 was up-regulated by ZEN at protein level. Meanwhile, the index of body length and offspring number of mutant nematodes was also higher than that of wild nematodes. These results showed that zearalenone inhibited the expression of collagen synthesis signaling pathway in the epidermis of C. elegans. 7. The results of microarray and RT-PCR were basically the same, indicating that 10 genes related to DAF-2 insulin-like signaling pathway and 5 downstream genes were affected by zearalenone, among them, daf-2, age-1, akt-1, a. Kt-2, sgk-1, pdk-1, daf-16, dct-15, daf-21 and ins-11 genes were up-regulated, while daf-18, hsp-1, hsf-1, dao-5 and egl-4 genes were down-regulated. DAF-16 # GFP was expressed in the nucleus and cytoplasm of all tissues and cells of the parasite. The nucleus enrichment of DAF-16 # GFP in the nematodes exposed to enone was significantly inhibited, and the release of DAF-16 # GFP from the nucleus of the nematodes exposed to enone was also significantly inhibited during the recovery process after heat stimulation. These results indicated that zearalenone induced up-regulation of DAF-2 insulin-like signaling pathway in C. elegans. The results of this study showed that zearalenone could affect the proliferation, developmental toxicity and neurotoxicity of nematodes by affecting the epidermal collagen synthesis pathway and DAF-2 insulin-like letter. The normal expression of zearalenone signaling pathway provides a scientific basis for further study on the mechanism of zearalenone toxicity regulation.
【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TS201.6
本文编号：2214453
[Abstract]:Mycotoxin is a kind of toxic secondary metabolites produced by fungi. It can cause cancer, infertility and other symptoms by contaminating food into humans and animals. It not only causes economic losses but also endangers human health. In recent years, there are many reports on mycotoxin toxicity, but also the organisms among model organisms. There are differences in accessibility and bioavailability, resulting in conflicting reports on toxic effects and toxicological mechanisms of mycotoxins among different model organisms. Compared with other model organisms, C. elegans is a simple model organism with a conservative genetic model that avoids individual differences and is conducive to standardization of toxicity assessment. Caenorhabditis elegans has become the first choice to evaluate the toxicity and toxicological mechanism of exogenous toxins. Therefore, the toxicity evaluation model of mycotoxins, especially zearalenone, was established to observe the toxic effect of mycotoxins on Caenorhabditis elegans, select the appropriate evaluation index and compare the corn red. The effects of mycophenone on gene expression profiles of Caenorhabditis elegans were investigated to explore the molecular toxicity mechanism of zearalenone on Caenorhabditis elegans. Specific research contents and results were as follows: 1. Toxicity evaluation model of Caenorhabditis elegans was established by using acute poisoning mortality, body length, offspring number and longevity as evaluation indicators. Toxic effects of aflatoxin B1, deoxynivalenol, fumonisin, T-2 toxin and zearalenone on Caenorhabditis elegans were studied. The results showed that the five mycotoxins had significant inhibitory effects on nematode development, reproductive capacity and longevity, and significantly reduced the length, width and number of offspring of adult nematodes. Aflatoxin B1 was the most sensitive toxin to reproductive indicators, followed by fumonisin and zearalenone. Comprehensive life span index was the most sensitive indicator to mycotoxins. 2. DIC microscope was used for zearalenone-infected nematodes. The results showed that the gonadal arms of nematodes in the exposed group atrophied significantly, the number and size of oocytes were significantly inhibited by zearalenone, and the generation time of nematodes was significantly prolonged. Zearalenone significantly reduced the resistance of C. elegans to heat stimulation and the survival rate of C. elegans after exposure to heat. Moreover, the behavioral ability of C. elegans was significantly affected by zearalenone, and the frequency of head swing and body bending significantly decreased after exposure for more than 48 hours. The results showed that zearalenone could weaken the resistance of C. elegans to external stimuli and cause serious behavioral defects, which had neurotoxic effects. 4. The growth and reproductive defects of C. elegans were observed in the adult worms after parental exposure to zearalenone. In the next generation of nematodes, only a limited recovery of developmental defects and no significant recovery of reproductive defects were observed. In addition, the toxic effect of the third generation of nematodes exposed to zearalenone was not continuous increase or decrease, but the most serious toxic effect of the second generation of nematodes, indicating that nematodes had the most serious toxic effect on zearalenone. These results indicate that zearalenone has teratogenic toxicity. 5. To further elucidate the mechanism of zearalenone's toxicity to Caenorhabditis elegans, microarray technique was used to analyze and compare the differences in gene expression of nematodes. There were 171,245,3149 abnormally differentially expressed genes in C. elegans after poisoning (2 times and more than that in the control group). These genes were mainly involved in signal transduction of phosphorylated cells, material metabolism, embryonic development, proliferation and differentiation of nerve cells, regulation of life span and behavior regulation by GO analysis. Gene analysis showed that zearalenone had developmental toxicity, reproductive toxicity, cytotoxicity and neurotoxicity. 6. Microarray and RT-PCR results showed that dpy-17, col-121, hch-1 were the epithelial collagen synthesis signaling pathways. The fluorescence expression of DPY-31 # GFP-labeled nematodes was significantly higher than that of normal nematodes, and the expression of DPY-31 was up-regulated by ZEN at protein level. Meanwhile, the index of body length and offspring number of mutant nematodes was also higher than that of wild nematodes. These results showed that zearalenone inhibited the expression of collagen synthesis signaling pathway in the epidermis of C. elegans. 7. The results of microarray and RT-PCR were basically the same, indicating that 10 genes related to DAF-2 insulin-like signaling pathway and 5 downstream genes were affected by zearalenone, among them, daf-2, age-1, akt-1, a. Kt-2, sgk-1, pdk-1, daf-16, dct-15, daf-21 and ins-11 genes were up-regulated, while daf-18, hsp-1, hsf-1, dao-5 and egl-4 genes were down-regulated. DAF-16 # GFP was expressed in the nucleus and cytoplasm of all tissues and cells of the parasite. The nucleus enrichment of DAF-16 # GFP in the nematodes exposed to enone was significantly inhibited, and the release of DAF-16 # GFP from the nucleus of the nematodes exposed to enone was also significantly inhibited during the recovery process after heat stimulation. These results indicated that zearalenone induced up-regulation of DAF-2 insulin-like signaling pathway in C. elegans. The results of this study showed that zearalenone could affect the proliferation, developmental toxicity and neurotoxicity of nematodes by affecting the epidermal collagen synthesis pathway and DAF-2 insulin-like letter. The normal expression of zearalenone signaling pathway provides a scientific basis for further study on the mechanism of zearalenone toxicity regulation.
【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TS201.6
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