牛磺酸对热应激致肉鸡主动脉内皮细胞损伤的保护作用研究

发布时间：2018-03-03 05:35

  本文选题：牛磺酸　切入点：热应激　出处：《沈阳农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：热应激是造成当前集约化养殖业经济严重损失的一种环境因素,对畜禽养殖业的长期发展影响巨大。很多动物实验证实,热应激导致中暑死亡的很大原因在于内皮细胞的大范围损伤。前人的研究发现,牛磺酸具有细胞保护作用,但牛磺酸对于热应激造成的内皮细胞损伤的保护作用机制还不清楚。因此,本研究探讨了牛磺酸对热应激肉鸡主动脉内皮细胞损伤的影响。以期为治疗或缓解肉鸡热应激相关疾病等提供理论依据。试验选取10日龄健康肉鸡,采用组织贴块法培养出原代肉鸡主动脉内皮细胞,用Ⅷ因子相关抗原试剂盒经免疫组化鉴定后,选用3-5代处在对数生长期的细胞进行后续试验,将内皮细胞随机分为五组,即正常对照(C)组、热应激(HS)组、热应激+低浓度Tau(HS+LTau)组、热应激+中浓度Tau(HS+MTau)组和热应激+高浓度Tau(HS+HTau)组,各组细胞均放入细胞培养箱(37℃,5%CO2)中培养24h,然后,正常对照组始终在37℃培养箱中培养,热应激各组细胞同时放在43℃恒温水浴锅中做热应激处理6h,再回细胞培养箱中复温培养1h,之后收集细胞和培养液上清进行各项指标的检测。结果如下:1)牛磺酸对HS肉鸡主动脉内皮细胞活性和凋亡率的影响:通过MTT比色法和AO/EB染色观察,发现,HS组肉鸡主动脉内皮细胞活性降低、凋亡率增加。培养液中添加Tau的内皮细胞相对成活率显著高于HS组(P0.05)、内皮细胞的凋亡率显著低于HS组(P0.05),结合MTT比色法与AO/EB染色结果的分析,表明牛磺酸对热应激导致的内皮细胞活性降低和凋亡率增加具有明显的抑制作用。其中,添加20mmol/L和40mmol/LTau的作用效果较好。2)牛磺酸对HS肉鸡主动脉内皮细胞氧化损伤的影响:与C组相比,HS组内皮细胞LDH活性和MDA含量极显著升高(P0.01),在抗氧化系统中,与C组相比,HS组内皮细胞T-SOD活性、GSH-Px活性以及T-AOC均极显著降低(P0.01);添加Tau后,与HS组相比,内皮细胞的LDH活性和MDA含量均显著降低(P0.05),内皮细胞的T-SOD活性、GSH-Px活性和T-AOC均显著升高(P0.05)。结果表明,HS导致内皮细胞中抗氧化能力的减弱,使内皮细胞处于严重的氧化损伤中,而添加Tau预防能降低内皮细胞中LDH活性和MDA含量,并提高抗氧化物酶SOD活性、GSH-Px活性和T-AOC,抑制了内皮细胞氧化损伤,增强了抗氧化能力,起到改善氧化损伤的效果。其中,添加40mmol/L Tau表现出较好的效果。3)牛磺酸对HS肉鸡主动脉内皮细胞凋亡相关基因和蛋白的影响:HS后,与C组相比,HS组内皮细胞中Bax、Caspase-9、Caspase-3、Cyt-c和P53等促凋亡因子的基因和蛋白表达水平显著升高(P0.05),抗凋亡因子Bcl-2基因和蛋白表达水平显著降低(P0.05);而添加Tau后,内皮细胞Bcl-2基因和蛋白表达水平与HS组相比显著提高(P0.05)、促凋亡因子Bax、Caspase-9、Caspase-3、Cyt-c和P53基因和蛋白表达水平与HS组相比均显著降低(P0.05)。综合基因和蛋白表达水平来看,HS诱导了促凋亡基因和蛋白的高表达,抑制了抗凋亡基因和蛋白的表达,造成内皮细胞大量凋亡,添加Tau可明显逆转这些变化,Tau通过提高抗凋亡因子Bcl-2的表达,降低促凋亡因子Bax、Caspase-9、Caspase-3、Cyt-c和P53的表达,来抑制线粒体介导的内皮细胞凋亡,发挥抗凋亡作用。其中,添加40mmol/LTau对HS诱导的内皮细胞凋亡的抑制作用效果较好。综上所述,添加一定浓度的Tau能够增强主动脉内皮细胞的抗氧化能力,抑制主动脉内皮细胞的凋亡。Tau可能通过抑制HS造成的主动脉内皮细胞抗氧化能力的降低和线粒体介导的凋亡通路的活化,发挥保护主动脉内皮细胞损伤的作用。整体比较来看,添加终浓度为40mmol/L的Tau的作用效果较好。
[Abstract]:Thermal stress is caused by an environmental factor in the current intensive breeding industry serious economic losses, the long-term development of the livestock industry a huge impact. Many animal experiments confirmed that the heat stress, resulting in a wide range of endothelial cell injury in the great cause of heatstroke death previous research found that taurine has cytoprotective effects, but the mechanism of protective effect of endothelial cell damage caused by taurine for heat stress is not clear. Therefore, this study investigated the effects of taurine on aorta endothelium in Heat Stressed Broilers. Treatment or alleviation of broiler heat stress related diseases in order to provide a theoretical basis for the test. The 10 day old healthy broilers, using tissue explant cultured method chicken aortic endothelial cells with factor VIII related antigen kit was identified by immunohistochemistry after the 3-5 generation in the logarithmic growth phase cells for subsequent Test, the endothelial cells were randomly divided into five groups: normal control group (C), heat stress (HS) group, heat stress and low concentration of Tau (HS+LTau) group, the concentration of Tau (HS+MTau) + heat stress group and the heat stress and high concentration of Tau (HS+HTau) group, each group of cells into cells incubator (at 37 5%CO2) in cultured 24h, then normal control group has 37 degrees in the incubator, the cells were placed in heat stress and constant temperature of 43 DEG C water bath heat stress 6h, then back to the cell culture box rewarming culture 1H detection after collecting cells and supernatant were the indexes. The results are as follows: 1) effect of taurine on HS activity and apoptosis rate of broiler aortic endothelial cells by MTT assay and AO/EB staining showed that HS group decreased broiler aortic endothelial cells, the apoptosis rate increased. The medium adding Tau endothelial cell relative survival rate was significantly higher than group HS (P0.05 ),鍐呯毊缁嗚優鐨勫噵浜＄巼鏄捐憲浣庝簬HS缁，

本文编号：1559762