本文選題：PRV + 細(xì)胞自噬　； 參考：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：偽狂犬病毒(Pseudorabies virus,PRV)是α皰疹病毒亞科的重要成員之一。偽狂犬病(Pseudorabies,PR)是由PRV感染引起,會導(dǎo)致懷孕母豬流產(chǎn)、呼吸和神經(jīng)系統(tǒng)障礙以及仔豬死亡。細(xì)胞自噬是機(jī)體維持內(nèi)環(huán)境穩(wěn)定的一種程序性死亡,機(jī)體利用自噬抵抗外來病原微生物入侵;此外,細(xì)胞自噬一般伴隨著細(xì)胞凋亡的發(fā)生,調(diào)控細(xì)胞凋亡的蛋白也可以調(diào)控細(xì)胞自噬。本研究在PRV感染PK15細(xì)胞中,利用免疫印跡(WB)方法,檢測PRV感染細(xì)胞的自噬水平;利用流式細(xì)胞術(shù)和WB方法,探討PRV感染細(xì)胞的凋亡水平;利用自噬抑制或誘導(dǎo)劑調(diào)控細(xì)胞自噬水平,探討自噬對PRV增殖的影響,利用RT-q PCR和WB檢測Beclin1、Bcl-2和Bcl-x L的轉(zhuǎn)錄和翻譯水平;最后利用免疫共沉淀實驗分析PRV感染PK15細(xì)胞中Beclin1與抗凋亡家族蛋白Bcl-2和Bcl-x L的結(jié)合水平。主要研究結(jié)果如下:1.PRV感染誘導(dǎo)細(xì)胞自噬,可能依賴于PI3K-Ⅰ/AKT信號通路PRV感染PK15細(xì)胞導(dǎo)致LC3-Ⅱ蛋白的表達(dá)明顯增多,表明PRV感染誘導(dǎo)了LC3-Ⅰ蛋白脂化成LC3-Ⅱ蛋白;且P62蛋白表達(dá)量也極顯著下降,進(jìn)一步表明PRV感染可以誘導(dǎo)細(xì)胞自噬。除此之外,還檢測了細(xì)胞自噬的主要信號通路PI3K-Ⅰ/AKT,結(jié)果顯示AKT蛋白表達(dá)水平無明顯變化,但其S473位點磷酸化水平顯著減少。2.PRV感染誘導(dǎo)細(xì)胞凋亡,同時激活Caspase-9和Caspase-8蛋白利用流式細(xì)胞術(shù)檢測了細(xì)胞凋亡,結(jié)果顯示PRV感染可誘導(dǎo)細(xì)胞凋亡;同時,利用了WB檢測PRV感染Caspase-9和Caspase-8剪切水平,結(jié)果表明Cleaved Caspase-9和Cleaved Caspase-8表達(dá)水平都顯著地上調(diào)。3.細(xì)胞自噬抑制PRV增殖分別利用自噬抑制劑3-MA和自噬誘導(dǎo)劑Rapa干擾自噬水平,探討其對病毒增殖的影響。研究結(jié)果表明,3-MA有效地抑制細(xì)胞自噬,PRV非結(jié)構(gòu)蛋白g E和TK基因m RNA水平、病毒衣殼蛋白VP26表達(dá)水平都顯著地增加;Rapa有效地誘導(dǎo)細(xì)胞自噬時,顯著地抑制病毒衣殼蛋白VP26表達(dá)水平。4.PRV感染抑制Beclin1和Bcl-2、Bcl-x L結(jié)合進(jìn)而促進(jìn)細(xì)胞自噬RT-q PCR和WB技術(shù)檢測結(jié)果表明,PRV感染自噬相關(guān)蛋白Beclin1的m RNA和蛋白水平顯著增加;Bcl-2的m RNA水平在感染6小時前呈現(xiàn)顯著上升、感染6小時后具有下降(12h、24h和36h)的趨勢,在蛋白水平呈現(xiàn)極顯著上升趨勢;Bcl-x L在m RNA水平呈現(xiàn)極顯著上升(6h、12h和24h)后下降(36h)的趨勢,在蛋白水平呈現(xiàn)極顯著上升趨勢。本研究利用免疫共沉淀的方法檢測了與Beclin1互作的豬宿主凋亡家族蛋白Bcl-2和Bcl-x L,研究結(jié)果表明PRV感染Beclin1能與Bcl-2和Bcl-x L結(jié)合,但結(jié)合的水平明顯減弱。
[Abstract]:Pseudorabies virus (PRV) is one of the important members of 偽 herpesvirus subfamily. Pseudorabies is caused by PRV infection, which can cause abortion, respiratory and nervous system disorders, and piglet death in pregnant sows. Autophagy is a programmed death of the body to maintain the stability of its internal environment. Autophagy is used by the body to resist the invasion of foreign pathogenic microorganisms. In addition, autophagy is usually accompanied by apoptosis. Proteins that regulate apoptosis also regulate autophagy. In this study, the level of autophagy in PK15 cells infected with PRV was detected by Western blotting method, and the apoptotic level of cells infected with PRV was studied by flow cytometry and WB methods. The effect of autophagy on PRV proliferation was studied by regulating autophagy level with autophagy inhibition or inducer. The transcription and translation levels of Bcl-2 and Bcl-x L in Beclin1 were detected by RT-q PCR and WB. Finally, the binding level of Beclin1 with anti-apoptotic family proteins Bcl-2 and Bcl-x L in PK15 cells infected with PRV was analyzed by immunoprecipitation assay. The main results were as follows: 1. The expression of LC3- 鈪，

本文編號：1933146