【摘要】：冷刺激作為寒區(qū)畜牧生產(chǎn)中常見的應(yīng)激原，易引起動物處于冷應(yīng)激狀態(tài)，從而導(dǎo)致畜產(chǎn)品質(zhì)量降低，動物死亡率增加，已成為制約我國養(yǎng)殖業(yè)快速發(fā)展的關(guān)鍵因素之一。冷應(yīng)激發(fā)生機(jī)制的研究受到國內(nèi)外科研工作者的廣泛關(guān)注。目前，對冷應(yīng)激發(fā)生機(jī)制的研究，往往只強(qiáng)調(diào)單個或少數(shù)幾個分子在應(yīng)激中的作用，缺乏從整體水平的系統(tǒng)性研究，從而難以準(zhǔn)確地評估動物是否真正處于應(yīng)激狀態(tài)。因此，本研究采用同重同位素相對與絕對定量（iTRAQ）結(jié)合質(zhì)譜技術(shù)篩選冷應(yīng)激大鼠血漿中的差異表達(dá)蛋白，以期在分子水平上尋找合適的冷應(yīng)激診斷標(biāo)志物，為冷應(yīng)激的防治提供理論依據(jù)。 本試驗采用12周齡、體重（340±20）g的健康SPF級雄性Wistar大鼠作為研究對象。將試驗鼠隨機(jī)分為常溫飼養(yǎng)組（Z組）和冷刺激組（L組）。常溫飼養(yǎng)溫度為（24.0±0.1）℃，冷刺激溫度為（4.0±0.1）℃，，冷刺激時間為12h。冷刺激后，采用ELISA、Western Blot和熒光定量PCR方法分別檢測血清中IL-2與IL-4、外周血淋巴細(xì)胞中HSP70和mRNA表達(dá)水平的變化，來判斷大鼠是否處于冷應(yīng)激狀態(tài)，從而確定冷應(yīng)激大鼠模型建立成功。根據(jù)冷應(yīng)激大鼠模型構(gòu)建的條件，開始正式冷刺激試驗。冷刺激后，采取大鼠血漿，采用iTRAQ技術(shù)篩選冷應(yīng)激大鼠血漿中的差異表達(dá)蛋白，通過COG注釋、GO和Pathway富集分析方法，找出與冷應(yīng)激反應(yīng)密切相關(guān)的差異表達(dá)蛋白；并經(jīng)Western Blot對其驗證。結(jié)果如下： 1.大鼠冷應(yīng)激模型的建立 與Z組相比，L組的大鼠血清中IL-2的含量顯著升高（P0.05），IL-4含量極顯著升高（P＜0.01）；外周血淋巴細(xì)胞內(nèi)HSP70及其mRNA表達(dá)量水平顯著升高（P0.05）。同時，結(jié)合本課題組成員的檢測結(jié)果：冷應(yīng)激大鼠血清中IL-6、TNF-α和CORT的含量極顯著升高（P＜0.01）、ACTH的含量顯著升高（P0.05）、IL-10無顯著變化。綜合表明，大鼠的冷應(yīng)激模型被成功構(gòu)建。 2.應(yīng)用iTRAQ技術(shù)篩選冷應(yīng)激大鼠血漿差異表達(dá)蛋白 共篩出39個差異表達(dá)蛋白，其中29個差異表達(dá)上調(diào)蛋白，10個差異表達(dá)下調(diào)蛋白。經(jīng)對差異表達(dá)蛋白的生物信息學(xué)分析，推測Ras相關(guān)蛋白Rap1b（Rap1b）和整合蛋白β-1（ITGB）與冷應(yīng)激反應(yīng)密切相關(guān)。 3.差異表達(dá)蛋白Rap1b和ITGB的Western Blot驗證 與Z組相比，L組的Rap1b相對表達(dá)水平呈顯著升高（P0.05）；而ITGB相對表達(dá)水平呈極顯著升高（P0.01）。 結(jié)論如下：本試驗成功構(gòu)建了大鼠冷應(yīng)激模型；應(yīng)用iTRAQ技術(shù)成功篩選出冷應(yīng)激大鼠血漿中的差異表達(dá)蛋白，經(jīng)過生物信息學(xué)分析后，找出了與大鼠冷應(yīng)激反應(yīng)密切相關(guān)的差異表達(dá)蛋白Rap1b和ITGB；經(jīng)Western Blot驗證，Rap1b和ITGB結(jié)果均為陽性，與iTRAQ技術(shù)檢測結(jié)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 鹵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 鹵0.1) 鈩

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