本文選題：脫氧雪腐鐮刀菌烯醇 + 細胞凋亡　； 參考：《福建農(nóng)林大學》2012年碩士論文

【摘要】：脫氧雪腐鐮刀菌烯醇（deoxynivalenol，DON）又名嘔吐毒素（vomitoxin，VT），是一種單端孢霉烯族類毒素，主要由某些鐮刀菌產(chǎn)生。在全世界范圍內(nèi)，糧食及其制品DON污染谷類的情況均非常普遍。這種毒素對人類的危害主要是通過人們直接食用谷物類食品或間接的攝入動物類食品（腎、肝臟、牛奶、雞蛋）所引起的。所有的動物對于DON的敏感程度是不同的，根據(jù)其敏感程度的不同的排序是豬㧐小鼠㧐大鼠㧐家禽㧐反芻動物。它不僅可以污染農(nóng)作物，還可以污染糧食制品，對人和動物可以產(chǎn)生廣泛的毒性效應，引起短期的惡心、嘔吐、腹瀉、腹痛、頭疼、頭暈和發(fā)燒。除了DON的一些急性作用外，研究表明長期暴露在DON中會造成實驗動物的體重減輕，食欲減退，營養(yǎng)吸收率降低和免疫失調(diào)。DON可以通過與60S核糖體結(jié)合而抑制多肽鏈的起始或延伸，，進而阻止蛋白質(zhì)的合成。DON對于粒性包細胞和一些生長較快的細胞有毒性作用，可以在細胞水平上誘導凋亡。DON誘導細胞凋亡和細胞毒作用是與MAPKs（SAPK/JNKs，P38MAPK，ERK1/2）的激活有關(guān)。根據(jù)DON的作用劑量、作用頻率、作用時間的不同、免疫功能的不同，DON會表現(xiàn)出相應的免疫激活或免疫抑制作用。這些發(fā)現(xiàn)為我們研究DON相關(guān)的疾病的分子機理提供了一定的幫助。 本研究主要分為兩個部分：一個是體外的DON對Hela和Chang liver細胞的毒理實驗；另一個是小鼠的體內(nèi)實驗。首先本研究運用細胞培養(yǎng)，流式細胞儀檢測，Western blot，Caspase3活性檢測等方法檢測了不同劑量的DON處理細胞不同時間后，觀察細胞的形態(tài)變化情況，檢測了細胞凋亡情況，檢測凋亡相關(guān)蛋白Caspase3的蛋白表達變化情況和活性變化情況；本實驗還通過克隆基因jnk，將其連接到熒光載體pEGFP-N1上來進行細胞定位，檢測JNK在DON誘導的細胞凋亡過程中的激活情況。在小鼠體內(nèi)實驗中，我們用提取的DON粗毒素來給小鼠腹腔注射，觀察注射組小鼠與對照組的小鼠比較的變化，檢測小鼠的體重變化情況，檢測小鼠的一些血液生化指標的變化和臟器的變化情況。本文探討分析了DON對于細胞凋亡的影響和對機體的毒性作用，初步揭示了DON的促凋亡作用和其分子機理。 本實驗獲得以下的實驗結(jié)果：①通過細胞形態(tài)學觀察，MTT的細胞增殖和細胞毒性的檢測以及流式細胞儀的凋亡細胞率的測定結(jié)果顯示，DON對于細胞具有明顯的促凋亡作用，并且這種作用與毒素的作用劑量和作用時間有關(guān)，DON的這種誘導凋亡的作用具有時間依賴性和劑量依賴性。②DON誘導細胞凋亡過程中激活了JNK信號轉(zhuǎn)導通路，JNK參與DON誘導的細胞凋亡的分子機制還需進一步證明。③DON誘導細胞凋亡過程中，Caspase3的活性顯著升高。④以禾谷鐮刀菌為菌株，經(jīng)過大米培養(yǎng)基的培養(yǎng)得到的DON粗毒素的提取液的濃度為3.273mg/mL。⑤DON會引起機體體重增加的減少，相應的一些生化指標發(fā)生變化。
[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.
【學位授予單位】：福建農(nóng)林大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：Q93;R378

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