HT-2毒素与玉米赤霉烯酮对小鼠卵母细胞的毒性影响及机制

发布时间：2018-07-07 17:30

本文选题：HT-2毒素 + ZEA　；参考：《南京农业大学》2016年博士论文

【摘要】：霉菌毒素是真菌或霉菌代谢产生的次级代谢产物,在植物,粮食和土壤中均可检测到霉菌毒素的存在。霉菌毒素分布广泛,在全世界各大洲均有霉菌毒素的发生和存在。随着全球贸易活动频繁,在粮食运输和贮藏过程中被霉菌毒素污染的可能性增加,从而造成饲料行业被霉菌毒素污染的情况更加严重。霉菌毒素已经被证实不仅会对实验动物和家畜产生各种毒性,同时也会影响人类健康。霉菌毒素对家畜和人类产生的毒性主要表现在影响免疫系统,消化系统,具有肝肾毒性,遗传毒性等。其中严重威胁家畜和人类健康的霉菌毒素主要包括玉米赤霉烯酮(ZEA ), T-2毒素和HT-2毒素,呕吐毒素,伏马毒素和黄曲霉毒素B1等。目前关于霉菌毒素毒性的研究很多,但是关于霉菌毒素对生殖方面的影响,尤其是对卵母细胞成熟和发育能力的影响以及相关毒性机制方面的研究较少。因此,本研究从体外和体内两个方面来探索霉菌毒素对小鼠卵母细胞成熟和发育能力的影响以及生物学机制。在体外,我们选取了 HT-2毒素和玉米赤霉烯酮,利用免疫荧光染色,免疫蛋白印记(western blot)和RT-PCR等方法来探索霉菌毒素对小鼠卵母细胞成熟和发育能力的影响,并且从细胞骨架,氧化应激,凋亡自噬和表观修饰四个方面来探索霉菌毒素的毒性机制。另外,我们又利用自然霉变的玉米配制饲料饲喂小鼠,进行体内试验,综合评价了霉菌毒素对小鼠卵母细胞的毒性及其机制。本研究共分为三个部分,主要的研究结果如下:—HT-2毒素影响小鼠卵母细胞成熟T-2毒素是A类单端孢霉菌毒素中毒性最强的一种毒素。目前已被证实会对家畜和人类产生各种毒性作用,它会诱导大脑,淋巴,造血和肠胃组织损伤等。HT-2毒素是T-2毒素的主要代谢产物。关于HT-2毒素对繁殖系统,尤其是卵母细胞成熟方面的研究还很少,因此在本试验中,我们主要探索了 HT-2毒素对卵母细胞成熟的影响及其相关机制。HT-2毒素会影响小鼠卵母细胞成熟,降低细胞膜和细胞质中微丝的表达,并且通过降低p-MAPK的蛋白水平从而破坏纺锤体构型。HT-2毒素也会诱导氧化应激和凋亡,具体表现在会导致ROS积聚,增加氧化应激相关基因Sod的mRNA水平,诱导早期凋亡,并且提高凋亡相关基因Caspase-3和Bax的mRNA水平。同时,HT-2毒素会提高自噬相关蛋白LC3和ATG12的表达水平和自噬相关基因Lc3和Atg14的表达,这意味着HT-2毒素会诱导小鼠卵母细胞自噬。除此之外,我们还检测了表观修饰方面。免疫荧光染色结果说明HT-2毒素会提高5mC的水平,而H3K9me2和H3K27me3的表达水平会下降,这些结果说明HT-2毒素会改变DNA甲基化和组蛋白甲基化水平。因此,我们的结果证明,HT-2毒素会通过影响细胞骨架,凋亡自噬,氧化应激和表观修饰等方面进而影响小鼠卵母细胞成熟。二玉米赤霉烯酮通过影响小鼠成熟卵子的表观修饰水平进而影响成熟卵子的发育能力ZEA是镰孢霉菌产生的霉菌毒素,已被证实会对家畜和人产生毒性。但是迄今为止关于ZEA影响小鼠卵子发育到胚胎的能力方面的研究较少。在本试验中我们发现,ZEA会影响小鼠卵子孤雌激活率,这说明ZEA会影响小鼠卵子发育到胚胎的能力。为了深入研究,我们从表观修饰方面探索ZEA影响小鼠卵子胚胎发育能力的可能机制。免疫荧光染色的结果分析说明ZEA会提高5mC水平,这意味着ZEA会提高小鼠卵子DNA甲基化水平。除此之外,组蛋白甲基化水平也发生了改变。高浓度ZEA处理组中卵子的H3K4me2, H3K9me3和H4K20me1, me2, me3水平显著下降。因此,我们的结果说明ZEA会通过影响表观修饰水平进而影响小鼠卵子胚胎发育能力。三霉菌毒素在体内环境中影响小鼠GV期卵母细胞表观修饰水平霉菌毒素普遍存在于谷物和饲料中。有研究报道,霉菌毒素会提高人肠细胞系的DNA甲基化水平。我们课题组先前的研究已经证明,霉菌毒素会影响小鼠卵母细胞的发育能力,在本实验中,我们从表观修饰方面来探索霉菌毒素体内降低小鼠卵母细胞发育能力的可能机制。试验选用自然霉变的玉米配制小鼠饲料,霉变玉米添加比例依次为0%,15%和30%,用该饲料饲喂小鼠四周。免疫荧光染色结果分析说明,高浓度的霉菌毒素会提高小鼠卵母细胞DNA甲基化水平。同时,组蛋白甲基化水平也发生了变化。其中,H3K9me3和H4K20me3水平显著升高,而H3K27me3和H4K20me2水平显著下降。因此,我们的结果说明,霉菌毒素会影响小鼠卵母细胞表观修饰水平,这可能是霉菌毒素降低小鼠卵母细胞发育能力的一个原因。
[Abstract]:Mycotoxins are secondary metabolites produced by the metabolism of fungi or moulds. Mycotoxins are detected in plants, grain and soil. Mycotoxins are widely distributed, and mycotoxins occur and exist in all continents around the world. With frequent Global trade activities, mycotoxins are contaminated by mycotoxins during grain transport and storage. The possibility of an increase in the possibility of contamination by mycotoxins in the feed industry is more serious. Mycotoxins have been proved not only to be toxic to experimental animals and livestock, but also to human health. The toxicity of mycotoxins to domestic animals and humans is now mainly affected by the immune system, the digestive system, and the liver. Renal toxicity, genetic toxicity, among which the major threat to domestic animal and human health mycotoxins mainly include ZEA, T-2 toxin and HT-2 toxin, vomit toxin, fumonoxin and aflatoxin B1. There are many studies on the toxicity of mycotoxins, but the effects of mycotoxins on reproductive aspects, especially in pairs. The effects of oocyte maturation and developmental ability and related toxicity mechanisms are rarely studied. Therefore, this study explored the effects of mycotoxins on the maturation and development of mouse oocytes and biological mechanisms from two aspects in vitro and in vivo. In vitro, we selected HT-2 toxin and Zea zearalenone, using immunization. Fluorescence staining, immunoglobulin imprint (Western blot) and RT-PCR were used to explore the effects of mycotoxins on the maturation and development of mouse oocytes, and to explore the toxic mechanism of mycotoxins from four aspects: cytoskeleton, oxidative stress, apoptosis autophagy and apparent modification. The toxicity and mechanism of mycotoxin to mouse oocytes were evaluated in vivo and in vivo. The results were divided into three parts. The main results were as follows: HT-2 toxin, which affects the mature T-2 toxin of oocyte in mice, is the most toxic toxin of class a monicillium toxin. Domestic animals and human beings have various toxic effects, which can induce.HT-2 toxins such as brain, lymph, hematopoiesis and intestinal tissue damage, which are the main metabolites of T-2 toxin. There are few studies on the reproductive system, especially oocyte maturation, with the HT-2 toxin. Therefore, in this experiment, we mainly explored the HT-2 toxin for oocyte formation. The effect of ripening and its related mechanism,.HT-2 toxin, can affect the maturation of mouse oocyte and decrease the expression of microfilament in the cell membrane and cytoplasm, and the destruction of the spindle configuration.HT-2 toxin by reducing the protein level of p-MAPK can also induce oxidative stress and apoptosis, which is manifested in the accumulation of ROS and the increase of the oxidative stress related gene Sod. MRNA levels, which induce early apoptosis, and increase the mRNA level of apoptosis related genes, Caspase-3 and Bax, and HT-2 toxin can increase the expression level of autophagy related protein LC3 and ATG12 and the expression of autophagy related genes Lc3 and Atg14, which means that HT-2 toxin can induce autophagy in mouse oocytes. Modification. The results of immunofluorescence staining showed that HT-2 toxin increased the level of 5mC, while the expression level of H3K9me2 and H3K27me3 decreased. These results indicated that HT-2 toxin would change the level of DNA methylation and histone methylation. Therefore, our results show that the HT-2 toxin can affect the cytoskeleton, apoptosis autophagy, oxidative stress and apparent expression. Modification and other aspects affect the maturation of mouse oocyte. Two zearalenone affects the development ability of mature oocytes by affecting the apparent modification level of mature oocytes in mice and further affecting the development ability of mature eggs, ZEA is a mycotoxin produced by Fusarium. It has been proved to be toxic to domestic animals and people. But so far, ZEA affects the development of mouse eggs. In this experiment, we found that ZEA could affect the rate of parthenogenetic activation in mouse oocytes, which indicates that ZEA affects the ability of mouse oocytes to develop into embryos. In order to further study, we explore the possible mechanism of ZEA affecting the fertility of mouse oocytes from the surface modification. The results showed that ZEA increased the level of 5mC, which means that ZEA could improve the level of DNA methylation in mouse eggs. In addition, the level of histone methylation also changed. The levels of H3K4me2, H3K9me3 and H4K20me1, me2, me3 in the high concentration ZEA treatment group decreased significantly. Therefore, our results indicate that ZEA will affect the apparent modified water. Three mycotoxins in the body environment affect the apparent modification of GV oocytes in mice. Mycotoxins generally exist in grain and feed. Studies have reported that mycotoxin can improve the level of DNA methylation in human intestinal cell lines. Previous studies in our group have proved that mycotoxin In this experiment, we explored the possible mechanism of reducing the development ability of mouse oocytes from the apparent modification in this experiment. The experiment selected the natural mouldy corn to prepare the mice feed. The proportion of mouldy corn added to the mice was 0%, 15% and 30%, and the mice were fed with this feed for four weeks. The results of immunofluorescence staining showed that high concentration of mycotoxin could increase the level of DNA methylation in mouse oocytes. At the same time, the level of histone methylation also changed. Among them, the level of H3K9me3 and H4K20me3 increased significantly, while the level of H3K27me3 and H4K20me2 decreased significantly. The apparent modification level of oocytes is probably one reason why mycotoxins reduce the ability of oocyte development in mice.
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S859.82

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