【作者】 王艳明；

【导师】 王振勇；

【作者基本信息】 山东农业大学 ， 临床兽医学， 2007， 硕士

【摘要】 为了进一步研究酒精阳性乳的发病机理,本研究从乳腺上皮细胞自由基代谢、膜组分改变和形态学变化的角度对酒精阳性乳的发病机理进行了探讨。检测了酒精阳性乳患牛乳腺上皮细胞自由基含量及相应抗氧化指标的变化。同时提取乳腺上皮细胞膜,检测了乳腺上皮细胞膜磷脂组分的相对百分含量,并对膜蛋白进行了SDS-PAGE电泳,蛋白质凝胶成像系统对电泳条带进行定性定量分析,对膜蛋白中具有重要功能的ATP酶的活性进行了测定。并取乳腺组织进行了常规病理切片和超微切片的观察。在调查酒精阳性乳发病原因和本实验室已有试验的基础上,自配复合药物“酸乳净”。把试验牛分为3组,一组作为阴性对照,另一组按照1包每头每天,连续5天投药作为治疗组,预防试验组在治疗组的基础上继续按1/4剂量投药,连续10天,检测其对产奶量和对酒精阳性乳的治疗作用。试验结果表明:1.已有的许多研究虽然表明酒精阳性乳是一种全身疾病的局部反应,但多数的研究只检测了其血液学指标的变化。本试验对比了阳性牛和阴性牛乳腺组织自由基及抗氧化指标的变化,表明阳性牛活性氧(ROS)含量、丙二醛(MDA)含量显著升高,而超氧化物歧化酶(SOD)的活性显著降低,谷胱甘肽过氧化物酶(GSH-Px)和总抗氧化能力(TAOC)略微降低,但与正常牛的差异不显著。乳腺组织自由基的增加是引起酒精阳性乳的启动因子,而抗氧化能力下降是引起发病的因素之一。2.乳腺上皮细胞膜SDS-PAGE电泳分离出5条主条带,与对照组相比,阳性牛蛋白Ⅰ带的相对百分含量显著升高,而蛋白Ⅱ和蛋白Ⅳ的相对百分含量显著降低,膜蛋白发生交联形成高聚物(HMP),乳腺上皮细胞膜磷脂酰丝氨酸(PS)、磷脂酰乙醇胺(PE)的相对百分含量明显下降。这是由于过多的自由基攻击生物膜系统,使乳腺组织细胞膜中膜蛋白组分、磷脂组分发生改变。3.自由基攻击膜蛋白使膜上具有重要生物学功能的蛋白质功能下降,特别是与能量和离子代谢有重要关系的ATP酶活性降低,同时磷脂组分的改变也使依赖膜脂而存在活性的ATP酶活性改变。4.细胞膜膜组分的改变造成细胞形态和功能的变化,表现为细胞发生肿胀、水泡变性,线粒体肿胀、嵴减少,内质网囊泡化,膜缺损、膜上微绒毛减少。说明酒精阳性乳由于乳腺上皮细胞膜膜组分及形态学的改变使细胞膜流动性和可变形性下降、通透性升高,乳汁分泌功能异常而分泌异常乳。5.与对照组相比,治疗组和预防试验组在投药后5天时,产奶量和抗氧化性能有较大提高,但15天时治疗组发生反弹,预防试验组性能继续提高,25天后治疗组和预防试验组都发生反弹,但预防试验组的性能较治疗前高,说明该药物可以用来预防治疗酒精阳性乳,但需要一定的剂量在饲料中维持较长时间添加。更多还原

【Abstract】 The changes of free radicals metabolism and membrane ingredients and morphology of mammary gland epithetical cells in cow suffered from Alcohol Positive Milk (APM) were studied in this paper for studying the molecular mechanism of APM/s occurring further. The concentrations of free radicals and anti-oxidation indexes were tested. Mammary gland epithetical cells were taken to analyze the phospholipids ingredients. SDS-Polyacrylamide gel electrophoresis was used for analyzing the membrane protein ingredients.ATPase activities which have important function in mammary protein were also tested. Mammary gland tissues were picked up to observe the conventional structure and ultrastructure. Based on the cause of alcohol pisitive milk(APM) and the research we have finished,we make up the compound drug“SUANRUJING”。The experimental cows in the study were devided into three groups,one group as negative group, the second group was fed“suanrujing”as treatment group, one package a day for 5 days while the third group continue to fed 1/4 for another 10 days on the basis of treatment group.The milk quantity and treatment were tested. The results show:1. Although many researches showed that APM is a systemic illness that reacts in part, previous researches only tested some changes in blood indexes. The study showed that the concentrations of ROS and MDA in cows suffered from APM increased significantly while the ability of anti-oxidation in the mammary gland itself is rather lower. Activities of GSH-Px and T-AOC go down appreciably, but not significantly, compared with the cow in control group.2. Five main bands were separated by SDS-Polyacrylamide gel electrophoresis. Compared with the control group, the comparative contents of bandⅠi ncrease significantly, while the comparative contents of bandⅡand bandⅣd ecrease significantly in the cow suffered from APM. The HMP increased, the result from the polymerization of membrane protein. Meanwhile, The comparative contents of the phospholipids ingredients decrease significantly, especially phosphatidylserine (PS) and phosphatidylethanolamine (PE). This may be resulted from the excess free radicals, which can attack the membrane, leading to the changes of protein and phospholipids ingredients in membrane.3. The protein activity with important biology function went dowm remarkly because of the free radicals’attack on membrane protein, especially ATPase activities related to energy and ions metabolism went down significantly. The changes of the phospholipids ingredients also can reduce the ATPase activities that depend on them.4. The changes of membrane ingredients induce the morphological and functional change. It appeared that the epithelial cells of mammary gland suffered blister denaturalization, mitochondria is intumescent, the number of the mitochondrial crests decrease, there were many vacuoles in the endoplasmic reticulum, membrane is absent, the microvilli on the membrane decrease. It was concluded that the produce of APM was due to the membrane ingredients changes and morphological changes, which made the fluidity and changeability of membrane decreased and its permeability disordered that leading to the secretion of APM.5. Compared with the control group, the milk quantity and the anti-oxygen function were improved significantly on the fifth day after the utility of drug, but the effect rebounded on the 15th day in treatment group while the prevention group went on improving. Both groups rebounded on the twentififth day, but the prevention group remained a higher porformence than the APM without treatmen.It was indicated that the drug can prenvent and treat the APM, but it needed to be addied for a long time.更多还原