【作者】 陈瑾；

【导师】 周小秋；

【作者基本信息】 四川农业大学 ， 动物营养与饲料科学， 2008， 硕士

【摘要】 本试验以原代培养鲤鱼肠上皮细胞为研究模型,研究了谷氨酰胺（Gln）对鲤鱼肠上皮细胞抗氧化能力的影响。研究共包括2个试验:试验一、体外培养鲤鱼IEC氧化应激模型的建立;试验二在试验一的基础上,应用已建立的肠上皮细胞氧化应激模型研究了Gln对氧化应激状态下IEC抗氧化能力的影响。试验一在原代培养的IEC培养基中分别添加0、15、30、45、60和100μM/L的过氧化氢（H₂O₂）溶液。结果表明:当H₂O₂浓度高于15μM/L时,对原代培养鲤鱼肠上皮细胞培养液乳酸脱氢酶（LDH）活力有极显著（P＜0.01）影响;当H₂O₂浓度高于45μM/L时,对原代培养鲤鱼肠上皮细胞培养液丙二醛（MDA）含量有极显著（P＜0.01）影响;且过氧化氢浓度与LDH活性和MDA含量呈极显著的正相关（r₁=+0.840,P＜0.01;r₂=+0.763,P＜0.01）。试验二共设6个处理:处理一,空白对照组,采用无血清培养基常规培养,不加处理因素;处理二,过氧化氢组,无血清培养基加入浓度为100μM/L过氧化氢;处理三至六,Gln处理组,含浓度分别为4、8、12和20mmol/LGln无血清培养基并加入终浓度为100μM/L过氧化氢。结果表明:鲤鱼肠上皮细胞用100μM/L H₂O₂处理后,细胞培养液中的LDH活性、MDA含量和细胞内蛋白羰基含量均极显著（P＜0.01）高于空白对照组;添加不同浓度的Gln能极显著（P＜0.01）降低培养液中的LDH活性、MDA含量和细胞内蛋白羰基含量,表明Gln能保护IEC细胞膜完整性,减少脂质和蛋白质氧化。鲤鱼肠上皮细胞用100μM/L H₂O₂处理后,碱性磷酸酶活性、钠钾ATP酶活性、γ-谷氨酰转移酶活性均极显著（P＜0.01）低于空白对照组;添加不同浓度的Gln能极显著（P＜0.01）提高碱性磷酸酶、钠钾ATP酶和γ-谷氨酰转移酶活性,表明Gln能抑制肠细胞功能受损。鲤鱼肠上皮细胞用100μM/L H₂O₂处理后,鲤鱼肠上皮细胞的抗超氧阴离子活力、抗羟自由基活力极显著降低（P＜0.01）;一氧化氮含量极显著升高（P＜0.01）;添加不同浓度的Gln能极显著（P＜0.01）提高细胞抗超氧阴离子活力、抗羟自由基活力,降低一氧化氮的含量。表明Gln能够提高细胞清除自由基的能力。鲤鱼肠上皮细胞用100μM/L H₂O₂处理后,超氧化物歧化酶活性、过氧化氢酶活性、谷胱甘肽过氧化酶活性、谷胱甘肽还原酶活性、谷胱甘肽S-转移酶活性、还原型谷胱甘肽含量和还原型谷胱甘肽与氧化型谷胱甘肽的比值极显著降低（P＜0.01）;添加不同浓度的Gln能极显著（P＜0.01）提高超氧化物歧化酶活性、过氧化氢酶活性、谷胱甘肽过氧化酶活性、谷胱甘肽还原酶活性、谷胱甘肽S-转移酶活性、还原型谷胱甘肽含量和还原型谷胱甘肽与氧化型谷胱甘肽的比值（P＜0.01）,表明Gln能够提高IEC抗氧化能力。结果说明:当过氧化氢含量为15-100μM/L时都能诱导鲤鱼肠上皮细胞发生氧化损伤。谷氨酰胺维护了鲤鱼肠上皮细胞结构的完整性,保持了细胞正常的分化能力和吸收功能。更多还原

【Abstract】 The present study explored the effects of glutamine on hydrogen peroxide （H₂O₂）-induced oxidative stress in isolated carp enterocytes. Firstly, in order to select an optimal H₂O₂ concentration to induce oxidative stress in enterocytes, cultures were treated with different concentrations of H₂O₂ （0-100μM） for 16 hours. The results showed that an increase of H₂O₂ concentrations beyond 15μM and 45μM enhanced LDH release and MDA levels in a dose-dependent manner, respectively （P＜0.01） . And there were significant correlations between glutamine content, lactate dehydrogenase activity and malondialdehyde levels in the culture medium （r₁=+0.840, P＜0.01; r₂=+0.763, P＜0.01）.Secondly, we then examined the cytoprotective effects of glutamine under conditions of oxidative stress. Cells were treated with glutamine （0-20 mmol/L） in the presence of H₂O₂ （100μM） for 16 hours. The control cells were kept in the glutamine-free MEM only. Results showed that glutamine completely blocked H₂O₂-stimulated release of lactate dehydrogenase （P＜0.01） . Furthermore glutamine reduced H₂O₂-induced increase in malondialdehyde levels and protein carbonyl content to levels seen in the control cultures, which means glutamine maintains cell membrane interity, preventing lipids and protein oxidation. Glutamine treatment completely prevented the decrease in alkaline phosphatase , sodium-potassium ATPase,γ- glutamyl transpeptidase induced by H₂O₂ （P＜0.01） , which means glutamine maintains cell function. In addition, glutamine treatment completely prevented the decrease in anti-superoxide anion activitity ,anti-hydroxy radical activitity and the increase in nitric oxide content by H₂O₂ （P＜0.01） , which means glutamine promoted cell antioxidant capacity by removing free radical. Glutamine treatment completely prevented the decrease in superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione S-transferase activities as well as reduced glutathione concentration and the ratio between reduced and oxidized glutathione induced by H₂O₂ （P＜0.01） ,which means glutamine enhances cell antioxidant capacity.In conclusion, hydrogen peroxide induced oxidative damage in IEC in a does-dependent manner. Glutamine completely prevents against H₂O₂-induced cytotoxicity in fish cells, due to its antioxidant potential. Thus, glutamine could maintain the cell structure and function during the conditions of oxidative stress.更多还原