本文關(guān)鍵詞： 禽呼腸孤病毒 自噬抑制劑 CQ E64d+pepstatinA 凋亡　出處：《揚(yáng)州大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：禽呼腸孤病毒(avian reovirus, ARV)感染可引起雞和火雞的病毒性關(guān)節(jié)炎、腱鞘炎和免疫抑制,給養(yǎng)禽業(yè)造成重大損失。已證實ARV感染能誘導(dǎo)細(xì)胞自噬和凋亡,且與病毒的致病作用有關(guān),但如何降低ARV誘導(dǎo)的細(xì)胞凋亡有待進(jìn)一步研究。本研究旨在探索自噬抑制劑CQ及E64d+pepstatinA對ARV誘導(dǎo)細(xì)胞凋亡的影響。首先使用MTT法檢測兩種自噬抑制劑對ARV誘導(dǎo)的細(xì)胞生長抑制的影響。結(jié)果顯示,經(jīng)兩種自噬抑制劑預(yù)處理的DF1細(xì)胞感染ARV后,病毒誘導(dǎo)的細(xì)胞生長抑制率顯著降低。進(jìn)一步經(jīng)形態(tài)學(xué)觀察發(fā)現(xiàn),兩種自噬抑制劑作用后,ARV誘導(dǎo)的DF1細(xì)胞融合病變顯著減少。最后,使用PI和Annexin V-FITC雙染法進(jìn)行流式細(xì)胞術(shù)檢測,結(jié)果顯示,經(jīng)CQ或E64d+pepstatinA處理后,ARV誘導(dǎo)的DFl細(xì)胞凋亡率顯著降低。Caspase-3的裂解水平高低通常被認(rèn)為是凋亡發(fā)生強(qiáng)弱的標(biāo)志。本實驗采用western blot法檢測ARV (GX/2010/1株)感染的DF1細(xì)胞caspase-3活性,發(fā)現(xiàn)經(jīng)CQ或E64d+pepstatinA預(yù)處理后,ARV誘導(dǎo)的DFl細(xì)胞凋亡率顯著降低。進(jìn)一步進(jìn)行病毒衣殼蛋白σ℃表達(dá)檢測及病毒滴度測定,發(fā)現(xiàn)兩種自噬抑制劑均可顯著降低病毒復(fù)制水平。禽呼腸孤病毒可利用凋亡使感染禽產(chǎn)生病理反應(yīng),鑒于兩種自噬抑制劑可在細(xì)胞水平抑制ARV誘導(dǎo)的細(xì)胞凋亡,進(jìn)一步進(jìn)行了動物實驗。使用CQ或E64d+pepstatinA對11日齡雞胚預(yù)處理后,接種ARV病毒(GX/2010/1株),并設(shè)立對照組。收集尿囊液進(jìn)行病毒滴度測定,發(fā)現(xiàn)經(jīng)兩種自噬抑制劑處理后,病毒滴度顯著低于單獨(dú)接毒組。收集組織樣品進(jìn)行蛋白檢測,并統(tǒng)計雞胚死亡率,繪制生存率曲線。結(jié)果顯示,CQ或E64d+pepstatinA均可顯著抑制ARV誘導(dǎo)的雞胚法氏囊、心臟及腸組織細(xì)胞凋亡,進(jìn)一步降低雞胚死亡率。本研究證明自噬抑制劑CQ和E64d+pepstatinA在抑制自噬的基礎(chǔ)上可顯著抑制ARV(GX/2010/1株)誘導(dǎo)的細(xì)胞凋亡,降低病毒復(fù)制水平；對雞胚,可顯著降低ARV(GX/2010/1株)導(dǎo)致的胚體死亡率。
[Abstract]:Avian reovirus virus (ARV) infection can cause viral arthritis, tenosynovitis and immunosuppression in chickens and turkeys. It has been proved that ARV infection can induce autophagy and apoptosis, and is related to the pathogenicity of the virus. However, how to reduce apoptosis induced by ARV needs further study. The purpose of this study is to explore the effect of autophagy inhibitor CQ and E64d pepstatinA on apoptosis induced by ARV. Firstly, MTT assay was used to detect the effect of two kinds of autophagy inhibitors on ARV induced apoptosis. The effect of cell growth inhibition. After two kinds of autophagy inhibitors pretreated DF1 cells infected with ARV, the inhibition rate of cell growth induced by virus was significantly decreased. After treatment with two autophagy inhibitors, the DF1 cell fusion lesion induced by ARV was significantly reduced. Finally, flow cytometry was performed with Pi and Annexin V-FITC staining, and the results showed that, The apoptosis rate of DFl cells induced by CQ or E64d pepstatinA was significantly decreased. The level of cleavage of Caspase-3 was generally regarded as a marker of the degree of apoptosis. The caspase-3 activity of DF1 cells infected with ARV GX / 2010 / 1 strain was detected by western blot assay. It was found that the apoptosis rate of DFl cells induced by CQ or E64d pepstatinA was significantly decreased, and the expression of virus capsid protein 蟽 鈩，

本文編號：1502857