脱氧雪腐镰刀菌烯醇诱导的细胞凋亡机理的初步研究

发布时间：2018-04-25 07:27

本文选题：脱氧雪腐镰刀菌烯醇 + 细胞凋亡　；参考：《福建农林大学》2012年硕士论文

【摘要】：脱氧雪腐镰刀菌烯醇（deoxynivalenol，DON）又名呕吐毒素（vomitoxin，VT），是一种单端孢霉烯族类毒素，主要由某些镰刀菌产生。在全世界范围内，粮食及其制品DON污染谷类的情况均非常普遍。这种毒素对人类的危害主要是通过人们直接食用谷物类食品或间接的摄入动物类食品（肾、肝脏、牛奶、鸡蛋）所引起的。所有的动物对于DON的敏感程度是不同的，根据其敏感程度的不同的排序是猪㧐小鼠㧐大鼠㧐家禽㧐反刍动物。它不仅可以污染农作物，还可以污染粮食制品，对人和动物可以产生广泛的毒性效应，引起短期的恶心、呕吐、腹泻、腹痛、头疼、头晕和发烧。除了DON的一些急性作用外，研究表明长期暴露在DON中会造成实验动物的体重减轻，食欲减退，营养吸收率降低和免疫失调。DON可以通过与60S核糖体结合而抑制多肽链的起始或延伸，，进而阻止蛋白质的合成。DON对于粒性包细胞和一些生长较快的细胞有毒性作用，可以在细胞水平上诱导凋亡。DON诱导细胞凋亡和细胞毒作用是与MAPKs（SAPK/JNKs，P38MAPK，ERK1/2）的激活有关。根据DON的作用剂量、作用频率、作用时间的不同、免疫功能的不同，DON会表现出相应的免疫激活或免疫抑制作用。这些发现为我们研究DON相关的疾病的分子机理提供了一定的帮助。本研究主要分为两个部分：一个是体外的DON对Hela和Chang liver细胞的毒理实验；另一个是小鼠的体内实验。首先本研究运用细胞培养，流式细胞仪检测，Western blot，Caspase3活性检测等方法检测了不同剂量的DON处理细胞不同时间后，观察细胞的形态变化情况，检测了细胞凋亡情况，检测凋亡相关蛋白Caspase3的蛋白表达变化情况和活性变化情况；本实验还通过克隆基因jnk，将其连接到荧光载体pEGFP-N1上来进行细胞定位，检测JNK在DON诱导的细胞凋亡过程中的激活情况。在小鼠体内实验中，我们用提取的DON粗毒素来给小鼠腹腔注射，观察注射组小鼠与对照组的小鼠比较的变化，检测小鼠的体重变化情况，检测小鼠的一些血液生化指标的变化和脏器的变化情况。本文探讨分析了DON对于细胞凋亡的影响和对机体的毒性作用，初步揭示了DON的促凋亡作用和其分子机理。本实验获得以下的实验结果：①通过细胞形态学观察，MTT的细胞增殖和细胞毒性的检测以及流式细胞仪的凋亡细胞率的测定结果显示，DON对于细胞具有明显的促凋亡作用，并且这种作用与毒素的作用剂量和作用时间有关，DON的这种诱导凋亡的作用具有时间依赖性和剂量依赖性。②DON诱导细胞凋亡过程中激活了JNK信号转导通路，JNK参与DON诱导的细胞凋亡的分子机制还需进一步证明。③DON诱导细胞凋亡过程中，Caspase3的活性显著升高。④以禾谷镰刀菌为菌株，经过大米培养基的培养得到的DON粗毒素的提取液的浓度为3.273mg/mL。⑤DON会引起机体体重增加的减少，相应的一些生化指标发生变化。
[Abstract]:Deoxynivalenolus deoxynivalenol (DON), also known as vomitoxin VTN, is a monosporin toxin, mainly produced by some Fusarium. DON contamination of grain and its products is common all over the world. The toxicity to humans is mainly caused by direct consumption of cereals or indirect intake of animal foods (kidney, liver, milk, eggs). The sensitivity of all animals to DON is different, according to the different order of sensitivity of pigs? Mice? Rats? Poultry? Ruminant. It can not only pollute crops, but also food products. It can have a wide range of toxic effects on humans and animals, causing short-term nausea, vomiting, diarrhea, abdominal pain, headache, dizziness and fever. In addition to some of the acute effects of DON, studies have shown that prolonged exposure to DON causes weight loss and loss of appetite in laboratory animals. Decreased nutrient absorption and immune maladjustment. Don can inhibit the initiation or extension of the polypeptide chain by binding to 60s ribosomes, thereby preventing protein synthesis. Don has toxic effects on granulocyte envelop cells and some fast-growing cells. The apoptosis and cytotoxicity induced by don at the cell level were related to the activation of MAPKsP38MAPKERK1 / 2. According to the different dose, frequency and time of action of DON, the different don of immune function will show the corresponding immune activation or immunosuppressive effect. These findings may be helpful for us to study the molecular mechanism of DON related diseases. This study is divided into two parts: one is the toxicity of DON to Hela and Chang liver cells in vitro, the other is in vivo experiment of mice. In this study, cell culture and flow cytometry were used to detect the Caspase3 activity of the cells treated with different doses of DON for different time. The morphologic changes and apoptosis of the cells were observed. The changes of protein expression and activity of apoptosis-related protein Caspase3 were detected, and the activation of JNK in the process of apoptosis induced by DON was detected by cloning the gene JNK and ligating it to the fluorescent vector pEGFP-N1 for cellular localization. In vivo experiments in mice, we used crude toxin extracted from DON to intraperitoneally inject mice to observe the changes of mice in the injection group compared with the control group, and to detect the changes of the body weight of the mice. The changes of some blood biochemical indexes and viscera of mice were detected. In this paper, the effects of DON on apoptosis and toxicity to organism were analyzed, and the mechanism of DON in promoting apoptosis and its molecular mechanism was preliminarily revealed. In this study, we obtained the following results: 1. The cell proliferation and cytotoxicity of MTT were observed by cell morphology. The results of flow cytometry showed that don had obvious apoptotic effect on cells. And this effect is related to the dose and time of action of toxin. The effect of don induced apoptosis is time dependent and dose dependent. 2DON activates the JNK signal transduction pathway and participates in the process of apoptosis. The molecular mechanism of apoptosis induced by DON should be further proved that the activity of Caspase3 increased significantly in the process of apoptosis induced by .3DON.4 Fusarium graminearum was the main strain. The concentration of DON crude toxin extracted from rice culture medium was 3.273mg/mL.5DON, which resulted in the loss of body weight and the change of some biochemical indexes.
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：Q93;R378

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