模式生物秀麗隱桿線蟲評(píng)價(jià)真菌毒素毒性毒理機(jī)制的研究
發(fā)布時(shí)間：2018-08-31 08:30
【摘要】：真菌毒素是一類由真菌產(chǎn)生的有毒次級(jí)代謝產(chǎn)物,通過污染食品進(jìn)入人類和動(dòng)物體內(nèi)引發(fā)癌癥、不孕等癥狀,有嚴(yán)重毒性,不僅造成經(jīng)濟(jì)損失還同時(shí)危害人類健康。近年來對于真菌毒素毒性的研究報(bào)道較多但由于模式生物個(gè)體之間生物可及性和生物利用率之間存在差異,造成真菌毒素在不同模式生物之間毒性效應(yīng)及毒理相關(guān)機(jī)制報(bào)道常有沖突。與其他模式生物比較模式生物秀麗隱桿線蟲身體構(gòu)造簡單、遺傳模式保守可避免個(gè)體差異有利于毒性評(píng)價(jià)的標(biāo)準(zhǔn)化。目前秀麗隱桿線蟲已經(jīng)成為評(píng)價(jià)外源性毒素毒性以及毒理機(jī)制的首選。因此本研究以秀麗隱桿線蟲為模式試驗(yàn)動(dòng)物,建立真菌毒素特別是玉米赤霉烯酮毒性評(píng)價(jià)模型,觀察真菌毒素對秀麗線蟲的毒性效應(yīng),選擇合適評(píng)價(jià)指標(biāo)并比較玉米赤霉烯酮對秀麗線蟲基因表達(dá)譜的影響,深入探討玉米赤霉烯酮對秀麗線蟲在分子水平的毒性作用機(jī)制。具體研究內(nèi)容及結(jié)果如下:1.采用急性中毒死亡率、身體長度、后代數(shù)目和最長壽命為評(píng)價(jià)指標(biāo)建立秀麗隱桿線蟲毒性評(píng)價(jià)模型,探究黃曲霉毒素B1、脫氧雪腐鐮刀菌烯醇、伏馬菌素、T-2毒素和玉米赤霉烯酮對秀麗線蟲的毒性效應(yīng)。結(jié)果顯示所用的五種真菌毒素對線蟲發(fā)育、生殖能力有顯著抑制作用并縮短線蟲壽命,可顯著降低線蟲成蟲的體長、體寬和后代數(shù)目。T-2毒素對于急性中毒死亡率指標(biāo)最為敏感,半數(shù)效應(yīng)劑量1.38 mg/L。黃曲霉毒素B1是對生殖指標(biāo)最敏感的的毒素,其次是伏馬菌素和玉米赤霉烯酮。綜合比較壽命指標(biāo)是線蟲對真菌毒素最為敏感的指標(biāo)。2.對玉米赤霉烯酮染毒線蟲使用DIC顯微鏡觀察,結(jié)果顯示染毒組線蟲性腺臂發(fā)生明顯萎縮,卵母細(xì)胞數(shù)目和大小均受玉米赤霉烯酮顯著抑制影響,線蟲世代時(shí)間顯著延長。且可見染毒組內(nèi)部分線蟲產(chǎn)卵器發(fā)育畸形,畸形比率與玉米赤霉烯酮存在劑量效應(yīng)關(guān)系,進(jìn)一步表明玉米赤霉烯酮存在嚴(yán)重生殖毒性效應(yīng)。3.在熱脅迫刺激下,玉米赤霉烯酮可顯著降低秀麗線蟲對熱刺激的抗性,降低受熱后線蟲存活幾率。并且染毒線蟲行為能力明顯受到玉米赤霉烯酮影響,暴露48 h以上線蟲頭部擺動(dòng)和身體彎曲頻率顯著降低。結(jié)果表明玉米赤霉烯酮可導(dǎo)致秀麗線蟲對外界刺激抗性減弱、行為能力產(chǎn)生嚴(yán)重缺陷,具有神經(jīng)毒性效應(yīng)。4.對秀麗線蟲采用親代染毒,后代斷毒恢復(fù)實(shí)驗(yàn),觀察實(shí)驗(yàn)結(jié)果顯示,玉米赤霉烯酮暴露導(dǎo)致的發(fā)育缺陷和生殖缺陷在線蟲世代間具有可傳遞性。在后代線蟲中,發(fā)育缺陷僅得到有限的恢復(fù),而生殖缺陷沒有得到明顯恢復(fù)。另外連續(xù)對三代線蟲暴露玉米赤霉烯酮,毒性效應(yīng)不是連續(xù)的增加或減弱,而是第二代線蟲產(chǎn)生最為嚴(yán)重的毒性效應(yīng),說明線蟲對玉米赤霉烯酮可產(chǎn)生一定代償作用。這些結(jié)果表明玉米赤霉烯酮具有一定致畸毒性效應(yīng)。5.為進(jìn)一步闡明玉米赤霉烯酮對秀麗線蟲毒性的相應(yīng)調(diào)控機(jī)制,采用Microarray技術(shù)分析比較線蟲基因表達(dá)的差異。結(jié)果顯示10、40和80 mg/L劑量玉米赤霉烯酮染毒后秀麗線蟲異常顯著差異表達(dá)基因數(shù)目分別為171、245、3149個(gè)(與對照組相差2倍及以上),這些基因通過GO分析功能集中在磷酸化細(xì)胞信號(hào)傳導(dǎo)、物質(zhì)代謝、胚胎發(fā)育、神經(jīng)細(xì)胞增殖分化、壽命調(diào)控和行為調(diào)控。統(tǒng)計(jì)分析受到顯著影響的信號(hào)通路有表皮膠原蛋白合成信號(hào)通路、Wnt-β-Catenin信號(hào)通路和DAF-2胰島素信號(hào)通路。基因分析結(jié)果顯示玉米赤霉烯酮具有發(fā)育毒性、生殖毒性、細(xì)胞毒性和神經(jīng)毒性。6.Microarray和RT-PCR結(jié)果顯示表皮膠原蛋白合成信號(hào)通路的dpy-17、col-121、hch-1明顯受到玉米赤霉烯酮抑制下調(diào),同時(shí)dpy-31和sqt-3基因被刺激上調(diào)。玉米赤霉烯酮染毒組DPY-31#GFP標(biāo)記線蟲的熒光表達(dá)量顯著高于正常線蟲,在蛋白水平表明ZEN上調(diào)DPY-31表達(dá)量。同時(shí)突變體線蟲對體長和后代數(shù)目評(píng)價(jià)指標(biāo)比野生型線蟲也更加敏感。這些結(jié)果顯示玉米赤霉烯酮抑制秀麗線蟲表皮膠原蛋白合成信號(hào)通路表達(dá)。7.Microarray結(jié)果和RT-PCR結(jié)果基本一致,顯示10個(gè)DAF-2 insulin-like信號(hào)通路的相關(guān)基因和5個(gè)通路下游基因受到玉米赤霉烯酮染毒影響,其中daf-2、age-1、akt-1、akt-2、sgk-1、pdk-1、daf-16、dct-15、daf-21和ins-11基因上調(diào),daf-18、hsp-1、hsf-1、dao-5和egl-4基因下調(diào)。DAF-16#GFP在線蟲各個(gè)組織細(xì)胞的細(xì)胞核和細(xì)胞質(zhì)中均有表達(dá)。在熱刺激條件下,正常組線蟲中的DAF-16#GFP迅速完成從細(xì)胞質(zhì)到細(xì)胞核的轉(zhuǎn)移但是玉米赤霉烯酮染毒組線蟲的DAF-16#GFP細(xì)胞核富集明顯受阻。并且在熱刺激后的恢復(fù)過程中,染毒組線蟲的DAF-16#GFP從細(xì)胞核的釋放過程也明顯受阻。這些結(jié)果顯示玉米赤霉烯酮誘導(dǎo)秀麗線蟲DAF-2 insulin-like信號(hào)通路表達(dá)上調(diào)。綜上所述,秀麗隱桿線蟲多個(gè)評(píng)價(jià)指標(biāo)對真菌毒素毒性作用敏感,是優(yōu)秀的評(píng)價(jià)真菌毒素毒性及其毒理機(jī)制的模式生物。同時(shí)本研究結(jié)果顯示玉米赤霉烯酮對于線蟲的生殖、發(fā)育毒性以及神經(jīng)毒性很可能是通過影響表皮膠原蛋白合成通路和DAF-2insulin-like信號(hào)通路的正常表達(dá)實(shí)現(xiàn),為進(jìn)一步探究玉米赤霉烯酮毒性調(diào)節(jié)機(jī)制提供了科學(xué)依據(jù)。
[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.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：TS201.6
本文編號(hào)：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.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：TS201.6
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