对虾和小鼠肝微粒体对T-2毒素体外转化的比较研究
发布时间：2018-11-16 17:21
【摘要】：T-2毒素被认为是A族单端孢霉烯族毒素中毒性最强的,由于其高毒性特性,曾被用于战争作为生化武器。某些真菌感染谷物后在适宜的条件下很容易产生真菌毒素。由于植物性成分在水产饲料中所占的比例不断增加,T-2毒素对水产动物的危害也在不断增加,这不但能造成水产品质量品质的下降,同时能带来严重的食品安全问题。因此,研究水产动物对T-2毒素的代谢显得至关重要。之前T-2毒素的研究主要集中在哺乳动物及家禽体内的代谢研究,而在水产动物中的研究较少。为此,深入研究对虾对T-2毒素的清除能力及代谢产物显得尤为重要。T-2毒素在动物中的代谢研究主要分为体内和体外,动物体内的研究主要是对T-2毒素在动物体内代谢后组织器官及排泄物中T-2毒素代谢产物的研究。而体外的研究主要运用动物的肝微粒体进行代谢,由于肝脏是动物的主要解毒器官,其中含有大量的解毒酶系,体外代谢的研究能为体内研究提供一定的参考。本论文主要运用对虾和小鼠肝微粒体体外代谢T-2毒素。同时对对虾和小鼠代谢T-2毒素的能力进行比较,期望找到对虾与小鼠代谢的差别,为进一步研究T-2毒素在对虾体内的代谢提供指导,也为水产饲料中T-2毒素限量标准的制定和风险评估提供参考。本论文分为四章内容,第一章是通过文献综述来主要介绍真菌毒素及其在不同动物体内的代谢研究,主要是T-2毒素在哺乳动物和家禽中的代谢。以及在植物中的代谢和动物中的代谢差别。第二章主要是制备对虾和小鼠肝微粒体,并检测肝微粒体中的蛋白,用对硝基苯酚法检测II相代谢酶UDPGT的活性。测得对虾肝微粒体中的蛋白含量是14.9 mg/g,小鼠肝微粒体中的蛋白含量是10.1 mg/g。对虾肝微粒体中的蛋白浓度高于小鼠。在对虾肝微粒体中几乎没有检测到UDPGT活性,而测得小鼠中的UDPGT酶活性为24.23 pmol/min-1mg-1。第三章是T-2-Glu A的制备及条件优化。利用对虾和小鼠肝微粒体制备T-2-GluA,但发现用对虾肝微粒体未合成出产物,可能是由于对虾和小鼠肝微粒体中所含的酶系的不同,可能是由于对虾肝微粒体中不存在UDPGT酶。用小鼠肝微粒体体外酶法合成T-2-GluA,并对合成条件进行了优化,肝微粒体蛋白浓度越大合成量越大,孵育时间90 min,曲拉通浓度为0.2%(w/v)时合成量最大。第四章用对虾和小鼠肝微粒体代谢T-2毒素,进行体外研究,T-2毒素在不同动物肝微粒体中的代谢存在明显差异,小鼠肝微粒体对T-2毒素的清除力是对虾肝微粒体的200多倍,说明对虾感染T-2毒素后在体内残留的时间也越久,对对虾造成的危害也越大。T-2毒素在对虾和小鼠肝微粒体中的代谢产物也不同,这主要是肝微粒体中代谢酶系的不同,水产动物与哺乳动物的代谢的差异都需要进一步研究。
[Abstract]:T-2 toxin is considered to be the most toxic in group A monotelene toxin, which has been used as a biological and chemical weapon in war because of its high toxicity. Some fungi can easily produce mycotoxins after they are infected with cereals under suitable conditions. Due to the increasing proportion of plant ingredients in aquatic feed, the harm of T-2 toxin to aquatic animals is also increasing, which can not only lead to the decline of quality of aquatic products, but also bring serious food safety problems. Therefore, it is very important to study the metabolism of T-2 toxin in aquatic animals. Previous studies of T-2 toxin were mainly focused on metabolism in mammals and poultry, but less in aquatic animals. Therefore, it is very important to study the scavenging ability and metabolites of T-2 toxin in prawn. The metabolism of T-2 toxin in animals is mainly divided into in vivo and in vitro. T-2 toxin metabolites in tissues, organs and excreta after metabolism of T-2 toxin in animals were studied in vivo. However, in vitro studies mainly use animal liver microsomes for metabolism. As the liver is the main detoxification organ of animals, which contains a large number of detoxification enzymes, the study of in vitro metabolism can provide a certain reference for in vivo research. In this paper, we used prawn and mouse liver microsome to metabolize T-2 toxin in vitro. At the same time, the ability of prawn and mice to metabolize T-2 toxin was compared, in order to find out the difference of metabolism between prawn and mouse, and to provide guidance for further study on metabolism of T-2 toxin in prawn. It also provides a reference for the formulation and risk assessment of the limit of T-2 toxin in aquatic feed. This paper is divided into four chapters. The first chapter is a review of the literature to introduce mycotoxins and their metabolism in different animals, mainly on the metabolism of T-2 toxin in mammals and poultry. And metabolic differences in plants and animals. In the second chapter, shrimp and mouse liver microsome were prepared, and the protein in liver microsome was detected. The activity of II metabolizing enzyme UDPGT was detected by p-nitrophenol method. The protein content in shrimp liver microsomes was determined to be 14. 9 mg/g, mouse liver microsomal protein content is 10. 1 mg/g.. The protein concentration in shrimp liver microsomes was higher than that in mice. Almost no UDPGT activity was detected in shrimp liver microsomes, while the activity of UDPGT enzyme in mice was 24.23 pmol/min-1mg-1.. The third chapter is about the preparation and optimization of T-2-Glu A. T-2-GluA was prepared from prawn and mouse liver microsomes, but it was found that the unsynthesized products from shrimp liver microsomes may be due to the different enzyme systems in shrimp and mouse liver microsomes. This may be due to the absence of UDPGT enzyme in shrimp liver microsomes. T-2-GluA was synthesized from mouse liver microsomes by enzymatic method in vitro, and the synthesis conditions were optimized. The higher the concentration of liver microsomal protein, the greater the amount of synthesis, and the maximum amount of synthesis was obtained when the incubation time was 90 min, when the concentration of Traitone was 0.2% (w / v). In chapter 4, the metabolism of T-2 toxin by prawn and mouse liver microsomes was studied in vitro. The metabolism of T-2 toxin in different animal liver microsomes was significantly different. The clearance of T-2 toxin by mouse liver microsomes was more than 200 times of that of prawn liver microsomes, which indicated that shrimp remained in vivo for a longer time after infection with T-2 toxin. The more harmful to prawns, the different metabolites of T-2 toxin in shrimp and mouse liver microsomes, which are mainly due to the different metabolic enzymes in liver microsomes. The differences in metabolism between aquatic animals and mammals require further study.
【学位授予单位】：广东海洋大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S945.1;S859.8
本文编号：2336140
[Abstract]:T-2 toxin is considered to be the most toxic in group A monotelene toxin, which has been used as a biological and chemical weapon in war because of its high toxicity. Some fungi can easily produce mycotoxins after they are infected with cereals under suitable conditions. Due to the increasing proportion of plant ingredients in aquatic feed, the harm of T-2 toxin to aquatic animals is also increasing, which can not only lead to the decline of quality of aquatic products, but also bring serious food safety problems. Therefore, it is very important to study the metabolism of T-2 toxin in aquatic animals. Previous studies of T-2 toxin were mainly focused on metabolism in mammals and poultry, but less in aquatic animals. Therefore, it is very important to study the scavenging ability and metabolites of T-2 toxin in prawn. The metabolism of T-2 toxin in animals is mainly divided into in vivo and in vitro. T-2 toxin metabolites in tissues, organs and excreta after metabolism of T-2 toxin in animals were studied in vivo. However, in vitro studies mainly use animal liver microsomes for metabolism. As the liver is the main detoxification organ of animals, which contains a large number of detoxification enzymes, the study of in vitro metabolism can provide a certain reference for in vivo research. In this paper, we used prawn and mouse liver microsome to metabolize T-2 toxin in vitro. At the same time, the ability of prawn and mice to metabolize T-2 toxin was compared, in order to find out the difference of metabolism between prawn and mouse, and to provide guidance for further study on metabolism of T-2 toxin in prawn. It also provides a reference for the formulation and risk assessment of the limit of T-2 toxin in aquatic feed. This paper is divided into four chapters. The first chapter is a review of the literature to introduce mycotoxins and their metabolism in different animals, mainly on the metabolism of T-2 toxin in mammals and poultry. And metabolic differences in plants and animals. In the second chapter, shrimp and mouse liver microsome were prepared, and the protein in liver microsome was detected. The activity of II metabolizing enzyme UDPGT was detected by p-nitrophenol method. The protein content in shrimp liver microsomes was determined to be 14. 9 mg/g, mouse liver microsomal protein content is 10. 1 mg/g.. The protein concentration in shrimp liver microsomes was higher than that in mice. Almost no UDPGT activity was detected in shrimp liver microsomes, while the activity of UDPGT enzyme in mice was 24.23 pmol/min-1mg-1.. The third chapter is about the preparation and optimization of T-2-Glu A. T-2-GluA was prepared from prawn and mouse liver microsomes, but it was found that the unsynthesized products from shrimp liver microsomes may be due to the different enzyme systems in shrimp and mouse liver microsomes. This may be due to the absence of UDPGT enzyme in shrimp liver microsomes. T-2-GluA was synthesized from mouse liver microsomes by enzymatic method in vitro, and the synthesis conditions were optimized. The higher the concentration of liver microsomal protein, the greater the amount of synthesis, and the maximum amount of synthesis was obtained when the incubation time was 90 min, when the concentration of Traitone was 0.2% (w / v). In chapter 4, the metabolism of T-2 toxin by prawn and mouse liver microsomes was studied in vitro. The metabolism of T-2 toxin in different animal liver microsomes was significantly different. The clearance of T-2 toxin by mouse liver microsomes was more than 200 times of that of prawn liver microsomes, which indicated that shrimp remained in vivo for a longer time after infection with T-2 toxin. The more harmful to prawns, the different metabolites of T-2 toxin in shrimp and mouse liver microsomes, which are mainly due to the different metabolic enzymes in liver microsomes. The differences in metabolism between aquatic animals and mammals require further study.
【学位授予单位】：广东海洋大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S945.1;S859.8
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