【作者】 王振勇；

【导师】 石发庆；

【作者基本信息】 东北农业大学 ， 临床兽医学， 2002， 博士

【摘要】 为了深入探讨雏鸡脑软化症发病的分子机理，本课题通过在采用低V_E饲料和添加过量UFA建立了雏鸡脑软化症实验模型。在进行临床观察、定期剖杀并检测体内V_E含量、体重和脑重的基础上，重点研究了与神经损伤有关的以下几个方面的内容：系统地检测了体内自由基代谢和抗氧化系统功能的变化；首次采用SDS-PAGE电泳法分离并检测了雏鸡脑软化症神经细胞膜蛋白组分，并以高效薄层层析法检测神经细胞膜磷脂组分；测定了脑软化症雏鸡神经细胞膜的膜流动性；首次系统检测了脑软化症雏鸡体内钙及微量元素含量，并进行了小脑组织总铁离子状态观察；采用酶组织化学方法系统地观察了脑组织中与脑能量代谢和神经递质有关的酶的活性；对相关组织进行了病理组织学和超微结构观察；并对脑组织中神经细胞的凋亡进行了检测。实验结果表明： 1．本实验通过采用低V_E饲料和添加过量UFA，成功复制出雏鸡脑软化模型，此方法简单易行，可比性强，为建立雏鸡脑软化模型提供了新的途径。并认为雏鸡站立时的脚爪蜷曲、爪尖着地、爪心悬空和小脑的严重出血、水肿、软化及小脑体积的明显增大是雏鸡脑软化的特异症状和剖检变化，可作为临床检查的主要依据，血清中V_E含量和XOD活性可作为实验室诊断指标。 2．饲料中V_E的缺乏和UFA的过量，使自由基的生成增多和清除减少，自由基在体内蓄积，表现为活性氧含量、MDA含量、NO含量的增加和XOD活性的增强。在体内自由基蓄积的同时，机体抗氧化系统功能出现代偿性增强，提高自由基的清除能力，以对抗自由基对机体的损伤，表现为GSH-Px活性和T-AOC的增强，但机体的代偿能力是有限的，发病雏鸡小脑中GSH-Px活性的突然降低和T-AOC的不再增高，表明GSH-Px在发病雏鸡小脑中存在耗竭现象。体内自由基含量增加是雏鸡脑软化的启动因子，小脑中GSH-Px的耗竭是雏鸡脑软化的因素之一。 3．自由基攻击生物膜系统，使神经细胞膜中膜蛋白组分、磷脂组分发生改变，导致膜流动性降低，神经细胞膜局部溶解破裂，导致神经细胞的变性和坏死。发病雏鸡脑神经细胞膜蛋白组分(高分子量蛋白增加)、磷脂组分(PE含量减少)的改变和膜流动性的降低是神经原变性和坏死的病理生理过程。 4．脑软化雏鸡体内铁、铜含量增高，锌含量降低，硒含量的变化与GSG-Px活性的变化相一致，而钙、钴、锰的含量没有变化。钙及微量元素含量的变化均与体内自由基的代谢有关。铁离子价态的变化表明铁在自由基的产生过程中起介导作用，对雏鸡脑软化症的发生起促进作用。发病小脑中特别是严重水肿区域亚铁离子的显色增强提示小脑软化灶的淡黄色可能是亚铁离子的颜色。 5．自由基对血管内皮细胞的攻击，使血管内皮细胞发生损伤，最终导致血管壁的破裂，是小脑出血和水肿的病理基础，同时血管内皮的损伤所致的血管内壁粗糙和PGI2和TXA2的合成紊乱是血管内凝血的根本原因。神经细胞细胞膜、线粒体和内质网的损伤及神经原的变性和坏死导致参与能量代谢和神经介质有关酶的活性减弱和局部脱失，使神经功能异常，从而使雏鸡出现运动障碍和神经症状。 博士学位论文 摘 要 东北农业大学 6 在雏鸡发生脑软化的过程中伴有神经细胞凋亡的现象，神经细胞凋亡的数量不仅受体内自由基含量的影响，同时也受到机体抗氧化系统功能的影响。更多还原

【Abstract】 The trial model with chicken encephalomalacia was set up by using the feed of low congtent of VE and supplying excessive unsaturated fatty acid in feed for studying the molecular mechanism of chicken encephalomalacia in the study. On the basis of observing the symptom and postmortem examination in clinic and determining the content of VE, the bodyweight, the weight of cerebrum and cerebellum, the focus of the study was on follows which were relative to neuropathic injury. The concentration of free radical and the function of antioxidant defense system of chicken with encephalomalacia were determined systematically. The protein and phospholipid constituent in membrane of neurocyte in brain of chicken with encephalomalacia was separated and studied with the method of SDS-PAGE electrophoresis and the method of high performance-thin layer chromatography respectively. The fluidity of neurocyte membrane of chicken with encephalomalacia was determined for the first time. The content of Ca and trace elements were determined in chicken encephalomalacia systematically and the status of Fe ion in cerebellum was observed. The activity of enzymes that were relative to metabolism of energy and neuro-medium in brain were checked systematically with the method of enzyme-histochemistry. Pathological examination was carried out by microscopic and ultramicroscopic observation. The apoptosis of neurocyte in brain was checked. Results and conclusion:1. The trial model with chicken encephalomalacia was set up successfully by using the feed of low content of VE and supplying excessive unsaturated fatty acid in feed in the study. The method was simple and practiced. It supplied the new way to set the trial model with encephalomalacia. The content of VE and the activity of XOD in serum caught be regarded as the diagnose index in laboratory.2. The deficiency of VE and the excessive UFA in feed resulted in the enhance of synthesizing and the reduce of clearing for free radical. So the free radical saved up in chicken. The content of ROS, MDA and NO and the activity of XOD increased. Meanwhile the function of antioxidant defense system increased to enhance the ability for clearing free radical and antagonized the damage to organism from free radical. So the activity of GSH-Px and T-AOC enhanced. But the compensative ability was limited and the activity of GSH-Px decreased abruptly and the T-AOC was not increased further in cerebellum of chicken with encephalomalacia. This showed that it existed the exhaustion of GSH-Px in cerebellum of chicken with encephalomalacia. The increase of free radical in organism was the primer and the exhaustion of GSH-Px in cerebellum was one of the factors for chicken encephalomalacia.3. The attack of free radical to organic membrane resulted in the changes of protein andphospholipid constituent in membrane of neurocyte and the decrease of fluidity of membrane of neurocyte. So the part of membrane of neurocyte solved and broke and the neurocyte became to degenerate and to necrosis. It was the patho-physiologic progress of neurocytic degeneration and necrosis that the change of protein and phospholipid constituent in neurocytic membrane and the decrease of fluidity of neurocytic membrane in chicken with encephalomalacia.4. The content of Fe and Cu increased and the content of Zn decreased, the change of the content of Se was consistent with the change of activity of GSH-Px and the content of Ca and Co did not changed in chicken with encephalomalacia. The change of Ca and trace element was all relative to the mechanism of free radical. The change of Fe ion status showed that Fe acted as medium in the progress of synthesizing free radical. It promoted the occurrence of chicken encephalomalacia. The coloring of Fe2+ increased in cerebellum of chicken with encephalomalacia, specially in district of edema. It pointed out that the light yellow-green color of soft district was the color of Fe2+.5. The attack of free radical to endotheliocyte in blood vessel resulted in the damage of endotheli更多还原