硒缺乏雏鸡肠粘膜差异蛋白的筛选验证及信号通路研究

发布时间：2018-02-16 14:55

  本文关键词： 硒缺乏 雏鸡 蛋白组学 肠粘膜 信号通路　出处：《黑龙江八一农垦大学》2016年博士论文　论文类型：学位论文

【摘要】：硒是人和动物体内重要的微量元素之一。硒缺乏导致幼龄动物腹泻,腹泻与肠粘膜免疫密切相关,而硒如何调控肠粘膜免疫尚不清楚。为了研究硒缺乏对雏鸡肠粘膜影响的分子机制,我们假设通过缺硒降低雏鸡十二指肠粘膜的抗氧化活性,增加核因子抑制剂激酶α(IKKα)的活性,并激活NF-κB通路,从而减弱免疫功能。本试验选择SPF雏鸡为试验动物,建立硒缺乏雏鸡模型,运用同位素标记相对和绝对定量-质谱联用技术(i TRAQ-LCMS/MS)定量鉴定硒缺乏雏鸡十二指肠粘膜差异蛋白质组。并使用生物信息学方法进行通路富集分析寻找差异代谢通路。采用Western Blot法验证十二指肠粘膜差异蛋白DNA拓扑异构酶β(TOPIIIβ)、蛋白酪氨酸激酶(PTK)、丝氨酸/苏氨酸激酶(AKT2),并分析NF-κB信号通路相关蛋白,P50,IKKα和双特异性促分裂原活化蛋白激酶(MEK2)。通过实时定量PCR(RT-Q-PCR)检测十二指肠粘膜差异蛋白TOPIIIβ、AKT2、PTK等m RNA的表达量。采用ELISA酶联免疫吸附法测定各段肠粘膜的分泌型免疫球蛋白A(SIg A)、十二指肠粘膜的氧化型谷胱甘肽(GSSG),谷胱甘肽(GSH)和谷胱甘肽过氧化物酶(GPx)的活性,并检测十二指肠粘膜的P65、NF-κB p65 DNA结合、p-IκB和IκB及炎性因子的活性。试验结果表明:1.与对照组相比,硒缺乏组血液及十二指肠粘膜组织中硒含量第7 d、21 d和35 d均降低,其中第7 d显著降低(p0.05),第21 d和35 d极显著降低(p0.01)。综合硒缺乏组雏鸡临床症状(精神萎靡,体重减轻和腹泻)和病理学检查(肌肉苍白,十二指肠出血和渗出性素质病)表明硒缺乏雏鸡模型成功建立。2.运用i TRAQ技术,识别和定量分析软件Mascot2.2和Proteome Discoverer1.4,按score1.2,或0.83(p0.05)Mascot筛选,最终发现硒缺乏雏鸡在7 d、21 d、35 d三个时间点,分别鉴定67种、127种、119种已知差异蛋白。在3个时间点共有25个差异蛋白具有表达一致性,其中有13个上调一致性蛋白,12个下调一致性蛋白。这25个差异蛋白KEGG通路分析富集在“Ras信号转导”等18个通路(p0.05)。3.在试验期第7 d、21 d、35 d,Western Blot结果显示,硒缺乏组雏鸡十二指肠粘膜TOPIIIβ蛋白在三个时间点的表达量分别是对照组的0.37倍、0.36倍、0.34倍;硒缺乏组AKT2蛋白分别是对照组的0.76倍、0.33倍、0.55倍;硒缺乏组PTK的表达量分别是对照组的1.12倍、0.89倍、2.16倍,验证结果与i TRAQ鉴定差异蛋白结果一致。硒缺乏组IKKα蛋白在三个时间点的表达量分别是对照组的2.16倍、2.10倍、2.31倍;在第7 d与21 d硒缺乏组MEK2的表达量分别是对照组的0.83倍与0.86倍,而在35 d硒缺乏组MEK2表达量是对照组的1.60倍;NF-κB(p50)表达在硒缺乏组显著增加(p0.05);这些参与NF-κB信号通路的硒缺乏差异蛋白被证实与i TRAQ鉴定结果一致。4.RT-Q-PCR结果显示,在7 d、21 d和35 d,硒缺乏组雏鸡十二指肠粘膜组织PTK表达高于对照组;硒缺乏组TOPIIIβ、AKT2、GPx1 m RNA表达水平低于对照组;MEK2 m RNA表达量在35d硒缺乏组增加(p0.05),这些基因表达水平与蛋白质组分析结果一致。5.ELISA结果显示,在7 d、21 d和35 d,硒缺乏引起雏鸡十二指肠粘膜SIg A分泌量显著降低(p0.05);在21 d和35 d,硒缺乏引起雏鸡空肠、回肠、盲肠和直肠粘膜中SIg A分泌量显著降低(p0.05)。并且硒缺乏导致雏鸡十二指肠粘膜GSSG活性升高,GSH和GPx活性减少(p0.05)。硒缺乏导致雏鸡十二指肠粘膜p-IκB、p65和NF-κB p65 DNA结合活性增高,IκB表达量降低(p0.01)。此外,硒缺乏增加雏鸡十二指肠粘膜促炎细胞因子肿瘤坏死因子-α、白介素-1β、干扰素γ和白介素-17A的表达量,抑制抗炎细胞因子转化生长因子(TGF-β1)和白介素-10的表达量。这说明硒缺乏通过激活雏鸡十二指肠粘膜NF-κB信号通路,降低免疫功能。综上所述,本试验在硒缺乏蛋白质组学研究基础上,证明了硒缺乏降低雏鸡十二指肠粘膜抗氧化活性及SIg A的分泌量,增加了IKKα和p50蛋白的表达量,并激活NF-κB信号通路,从而降低十二指肠粘膜免疫功能,促进炎症的发生。
[Abstract]:Selenium is one of the important trace elements in human and animal. In young animal selenium deficiency diarrhea, diarrhea and intestinal mucosal immunity is closely related, and selenium how to regulate intestinal mucosal immunity is unclear. In order to investigate the mechanism of effect of selenium deficiency on intestinal mucosa of chicks, we hypothesized that selenium deficiency decreased antioxidant activity by chick duodenal mucosa. The increase of nuclear factor inhibitor kinase alpha (IKK alpha) activity and activation of NF- B pathway, which weakens the immune function. This experiment with SPF as experimental animal, the establishment of selenium deficiency in chicks model, using isobaric tags for relative and absolute quantitative mass spectrometry (I TRAQ-LCMS/MS) quantitative identification of selenium deficiency in chicks of duodenal mucosa difference protein group. And the use of biological information pathway enrichment analysis for differences between the metabolic pathways methodology. By using Western Blot method to verify the duodenal mucosal protein DNA Topo beta (TOPIII beta), protein tyrosine kinase (PTK), serine / threonine kinase (AKT2), and analyze the NF- B signaling pathway related protein, P50, IKK alpha and dual specificity mitogen activated protein kinase (MEK2). By quantitative real-time PCR (RT-Q-PCR) detection of duodenal mucosal protein TOPIII beta. AKT2, PTK and M Expression of RNA. Determination of intestinal mucosa sections by ELISA ELISA method of secretory immunoglobulin A (SIg A), oxidized glutathione in the duodenal mucosa (GSSG), glutathione (GSH) and Gu Guanggan (GPx) glutathione peroxidase activity, and detection of duodenal mucosa P65 according to NF-, p65 kappa B DNA, p-I B and I kappa kappa B and inflammatory factor activity. The results showed that: 1. compared with the control group, blood group and duodenal mucosa in seventh D selenium content in selenium deficiency, 21 d and 35 d decreased seventh, D significantly decreased (P0.05), twenty-first D and 35 d significantly decreased (P0.01). The clinical symptoms of selenium deficiency group chickens (listlessness, weight loss and diarrhea) and pathology (pale muscle, duodenal bleeding and exudative diathesis) showed that selenium deficiency in chicken model was successfully established using.2. I TRAQ technology, identification and quantitative analysis software Mascot2.2 and Proteome according to Discoverer1.4, score1.2, or 0.83 (P0.05) Mascot screening, eventually found chickens at 7 d, 21 d selenium deficiency, 35 d three time points were identified 67 species, 127 species, 119 kinds of known proteins. A total of 3 time points 25 differential protein expression is consistent with 13 up 12 down regulated protein consistency, consistency of protein. The difference between the 25 protein KEGG pathway enrichment analysis in the Ras signal transduction pathway and 18 (P0.05).3. in the test period of seventh D, 21 d, 35 d, Western Blot results showed that selenium deficiency group were duodenal stick 鑶淭OPIII尾铔嬬櫧鍦ㄤ笁涓�鏃堕棿鐐圭殑琛ㄨ揪閲忓垎鍒�鏄�瀵圭収缁勭殻�

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