發(fā)布時(shí)間：2018-02-16 14:55

  本文關(guān)鍵詞： 硒缺乏 雛雞 蛋白組學(xué) 腸粘膜 信號(hào)通路　出處：《黑龍江八一農(nóng)墾大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：硒是人和動(dòng)物體內(nèi)重要的微量元素之一。硒缺乏導(dǎo)致幼齡動(dòng)物腹瀉,腹瀉與腸粘膜免疫密切相關(guān),而硒如何調(diào)控腸粘膜免疫尚不清楚。為了研究硒缺乏對(duì)雛雞腸粘膜影響的分子機(jī)制,我們假設(shè)通過缺硒降低雛雞十二指腸粘膜的抗氧化活性,增加核因子抑制劑激酶α(IKKα)的活性,并激活NF-κB通路,從而減弱免疫功能。本試驗(yàn)選擇SPF雛雞為試驗(yàn)動(dòng)物,建立硒缺乏雛雞模型,運(yùn)用同位素標(biāo)記相對(duì)和絕對(duì)定量-質(zhì)譜聯(lián)用技術(shù)(i TRAQ-LCMS/MS)定量鑒定硒缺乏雛雞十二指腸粘膜差異蛋白質(zhì)組。并使用生物信息學(xué)方法進(jìn)行通路富集分析尋找差異代謝通路。采用Western Blot法驗(yàn)證十二指腸粘膜差異蛋白DNA拓?fù)洚悩?gòu)酶β(TOPIIIβ)、蛋白酪氨酸激酶(PTK)、絲氨酸/蘇氨酸激酶(AKT2),并分析NF-κB信號(hào)通路相關(guān)蛋白,P50,IKKα和雙特異性促分裂原活化蛋白激酶(MEK2)。通過實(shí)時(shí)定量PCR(RT-Q-PCR)檢測(cè)十二指腸粘膜差異蛋白TOPIIIβ、AKT2、PTK等m RNA的表達(dá)量。采用ELISA酶聯(lián)免疫吸附法測(cè)定各段腸粘膜的分泌型免疫球蛋白A(SIg A)、十二指腸粘膜的氧化型谷胱甘肽(GSSG),谷胱甘肽(GSH)和谷胱甘肽過氧化物酶(GPx)的活性,并檢測(cè)十二指腸粘膜的P65、NF-κB p65 DNA結(jié)合、p-IκB和IκB及炎性因子的活性。試驗(yàn)結(jié)果表明:1.與對(duì)照組相比,硒缺乏組血液及十二指腸粘膜組織中硒含量第7 d、21 d和35 d均降低,其中第7 d顯著降低(p0.05),第21 d和35 d極顯著降低(p0.01)。綜合硒缺乏組雛雞臨床癥狀(精神萎靡,體重減輕和腹瀉)和病理學(xué)檢查(肌肉蒼白,十二指腸出血和滲出性素質(zhì)病)表明硒缺乏雛雞模型成功建立。2.運(yùn)用i TRAQ技術(shù),識(shí)別和定量分析軟件Mascot2.2和Proteome Discoverer1.4,按score1.2,或0.83(p0.05)Mascot篩選,最終發(fā)現(xiàn)硒缺乏雛雞在7 d、21 d、35 d三個(gè)時(shí)間點(diǎn),分別鑒定67種、127種、119種已知差異蛋白。在3個(gè)時(shí)間點(diǎn)共有25個(gè)差異蛋白具有表達(dá)一致性,其中有13個(gè)上調(diào)一致性蛋白,12個(gè)下調(diào)一致性蛋白。這25個(gè)差異蛋白KEGG通路分析富集在“Ras信號(hào)轉(zhuǎn)導(dǎo)”等18個(gè)通路(p0.05)。3.在試驗(yàn)期第7 d、21 d、35 d,Western Blot結(jié)果顯示,硒缺乏組雛雞十二指腸粘膜TOPIIIβ蛋白在三個(gè)時(shí)間點(diǎn)的表達(dá)量分別是對(duì)照組的0.37倍、0.36倍、0.34倍;硒缺乏組AKT2蛋白分別是對(duì)照組的0.76倍、0.33倍、0.55倍;硒缺乏組PTK的表達(dá)量分別是對(duì)照組的1.12倍、0.89倍、2.16倍,驗(yàn)證結(jié)果與i TRAQ鑒定差異蛋白結(jié)果一致。硒缺乏組IKKα蛋白在三個(gè)時(shí)間點(diǎn)的表達(dá)量分別是對(duì)照組的2.16倍、2.10倍、2.31倍;在第7 d與21 d硒缺乏組MEK2的表達(dá)量分別是對(duì)照組的0.83倍與0.86倍,而在35 d硒缺乏組MEK2表達(dá)量是對(duì)照組的1.60倍;NF-κB(p50)表達(dá)在硒缺乏組顯著增加(p0.05);這些參與NF-κB信號(hào)通路的硒缺乏差異蛋白被證實(shí)與i TRAQ鑒定結(jié)果一致。4.RT-Q-PCR結(jié)果顯示,在7 d、21 d和35 d,硒缺乏組雛雞十二指腸粘膜組織PTK表達(dá)高于對(duì)照組;硒缺乏組TOPIIIβ、AKT2、GPx1 m RNA表達(dá)水平低于對(duì)照組;MEK2 m RNA表達(dá)量在35d硒缺乏組增加(p0.05),這些基因表達(dá)水平與蛋白質(zhì)組分析結(jié)果一致。5.ELISA結(jié)果顯示,在7 d、21 d和35 d,硒缺乏引起雛雞十二指腸粘膜SIg A分泌量顯著降低(p0.05);在21 d和35 d,硒缺乏引起雛雞空腸、回腸、盲腸和直腸粘膜中SIg A分泌量顯著降低(p0.05)。并且硒缺乏導(dǎo)致雛雞十二指腸粘膜GSSG活性升高,GSH和GPx活性減少(p0.05)。硒缺乏導(dǎo)致雛雞十二指腸粘膜p-IκB、p65和NF-κB p65 DNA結(jié)合活性增高,IκB表達(dá)量降低(p0.01)。此外,硒缺乏增加雛雞十二指腸粘膜促炎細(xì)胞因子腫瘤壞死因子-α、白介素-1β、干擾素γ和白介素-17A的表達(dá)量,抑制抗炎細(xì)胞因子轉(zhuǎn)化生長(zhǎng)因子(TGF-β1)和白介素-10的表達(dá)量。這說明硒缺乏通過激活雛雞十二指腸粘膜NF-κB信號(hào)通路,降低免疫功能。綜上所述,本試驗(yàn)在硒缺乏蛋白質(zhì)組學(xué)研究基礎(chǔ)上,證明了硒缺乏降低雛雞十二指腸粘膜抗氧化活性及SIg A的分泌量,增加了IKKα和p50蛋白的表達(dá)量,并激活NF-κB信號(hào)通路,從而降低十二指腸粘膜免疫功能,促進(jìn)炎癥的發(fā)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椂闂寸偣鐨勮〃杈鵑噺鍒嗗埆鏄鐓х粍鐨，

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