冷应激大鼠血浆差异表达蛋白的筛选与鉴定
发布时间：2018-07-28 07:40
【摘要】：冷刺激作为寒区畜牧生产中常见的应激原，易引起动物处于冷应激状态，从而导致畜产品质量降低，动物死亡率增加，已成为制约我国养殖业快速发展的关键因素之一。冷应激发生机制的研究受到国内外科研工作者的广泛关注。目前，对冷应激发生机制的研究，往往只强调单个或少数几个分子在应激中的作用，缺乏从整体水平的系统性研究，从而难以准确地评估动物是否真正处于应激状态。因此，本研究采用同重同位素相对与绝对定量（iTRAQ）结合质谱技术筛选冷应激大鼠血浆中的差异表达蛋白，以期在分子水平上寻找合适的冷应激诊断标志物，为冷应激的防治提供理论依据。 本试验采用12周龄、体重（340±20）g的健康SPF级雄性Wistar大鼠作为研究对象。将试验鼠随机分为常温饲养组（Z组）和冷刺激组（L组）。常温饲养温度为（24.0±0.1）℃，冷刺激温度为（4.0±0.1）℃，，冷刺激时间为12h。冷刺激后，采用ELISA、Western Blot和荧光定量PCR方法分别检测血清中IL-2与IL-4、外周血淋巴细胞中HSP70和mRNA表达水平的变化，来判断大鼠是否处于冷应激状态，从而确定冷应激大鼠模型建立成功。根据冷应激大鼠模型构建的条件，开始正式冷刺激试验。冷刺激后，采取大鼠血浆，采用iTRAQ技术筛选冷应激大鼠血浆中的差异表达蛋白，通过COG注释、GO和Pathway富集分析方法，找出与冷应激反应密切相关的差异表达蛋白；并经Western Blot对其验证。结果如下： 1.大鼠冷应激模型的建立 与Z组相比，L组的大鼠血清中IL-2的含量显著升高（P0.05），IL-4含量极显著升高（P＜0.01）；外周血淋巴细胞内HSP70及其mRNA表达量水平显著升高（P0.05）。同时，结合本课题组成员的检测结果：冷应激大鼠血清中IL-6、TNF-α和CORT的含量极显著升高（P＜0.01）、ACTH的含量显著升高（P0.05）、IL-10无显著变化。综合表明，大鼠的冷应激模型被成功构建。 2.应用iTRAQ技术筛选冷应激大鼠血浆差异表达蛋白 共筛出39个差异表达蛋白，其中29个差异表达上调蛋白，10个差异表达下调蛋白。经对差异表达蛋白的生物信息学分析，推测Ras相关蛋白Rap1b（Rap1b）和整合蛋白β-1（ITGB）与冷应激反应密切相关。 3.差异表达蛋白Rap1b和ITGB的Western Blot验证 与Z组相比，L组的Rap1b相对表达水平呈显著升高（P0.05）；而ITGB相对表达水平呈极显著升高（P0.01）。 结论如下：本试验成功构建了大鼠冷应激模型；应用iTRAQ技术成功筛选出冷应激大鼠血浆中的差异表达蛋白，经过生物信息学分析后，找出了与大鼠冷应激反应密切相关的差异表达蛋白Rap1b和ITGB；经Western Blot验证，Rap1b和ITGB结果均为阳性，与iTRAQ技术检测结果相一致。
[Abstract]:Cold stimulation, as a common stressor in livestock production in cold regions, is easy to cause animals to be in cold stress state, which leads to the decrease of animal product quality and the increase of animal mortality, which has become one of the key factors restricting the rapid development of animal husbandry in China. The research on the mechanism of cold stress has been paid much attention by researchers at home and abroad. At present, the research on the mechanism of cold stress often emphasizes the role of single or a few molecules in stress, and lacks systematic research on the whole level, so it is difficult to accurately assess whether animals are really in stress state. Therefore, the differential expression proteins in cold stress rat plasma were screened by using the technique of iso-isotope and absolute quantitative (iTRAQ) combined with mass spectrometry, in order to find suitable diagnostic markers of cold stress at molecular level. To provide a theoretical basis for the prevention and treatment of cold stress. Healthy SPF grade male Wistar rats of 12 weeks old and weight of (340 卤20) g were used as the study objects. The rats were randomly divided into normal temperature feeding group (Z group) and cold stimulation group (L group). The feeding temperature at room temperature was (24.0 卤0.1) 鈩
本文编号：2149385
[Abstract]:Cold stimulation, as a common stressor in livestock production in cold regions, is easy to cause animals to be in cold stress state, which leads to the decrease of animal product quality and the increase of animal mortality, which has become one of the key factors restricting the rapid development of animal husbandry in China. The research on the mechanism of cold stress has been paid much attention by researchers at home and abroad. At present, the research on the mechanism of cold stress often emphasizes the role of single or a few molecules in stress, and lacks systematic research on the whole level, so it is difficult to accurately assess whether animals are really in stress state. Therefore, the differential expression proteins in cold stress rat plasma were screened by using the technique of iso-isotope and absolute quantitative (iTRAQ) combined with mass spectrometry, in order to find suitable diagnostic markers of cold stress at molecular level. To provide a theoretical basis for the prevention and treatment of cold stress. Healthy SPF grade male Wistar rats of 12 weeks old and weight of (340 卤20) g were used as the study objects. The rats were randomly divided into normal temperature feeding group (Z group) and cold stimulation group (L group). The feeding temperature at room temperature was (24.0 卤0.1) 鈩
本文编号：2149385
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