本文關(guān)鍵詞：鯉魚TLRs功能及鯉春病毒血癥病毒感染后免疫相關(guān)基因應(yīng)激表達(dá)機(jī)制的研究　出處：《上海海洋大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 鯉魚 Toll樣受體 鯉春病毒血癥病毒 免疫相關(guān)基因 表達(dá)機(jī)制

【摘要】：Toll樣受體(Toll-like receptors,TLRs)是識(shí)別病毒病原相關(guān)分子模式(Pattern associated molecular patterns,PAMPs)的主要模式識(shí)別受體(Pattern recognition receptors,PRRs),其識(shí)別病毒PAMPs后,通過依賴My D88(Myeloid differentiation factor 88,My D88)或TRIF(TIR domain-containing adaptor-inducing interferon-β,TRIF)信號(hào)傳導(dǎo)通路,誘導(dǎo)核因子κB(Nuclear factor-κB,NF-κB)和干擾素調(diào)節(jié)因子(Interferon regulatory factors,IRFs)的表達(dá),啟動(dòng)固有免疫和適應(yīng)性免疫而抵御病毒入侵。至今在硬骨魚類中已鑒定了20種TLRs,大多數(shù)魚類TLRs能在哺乳動(dòng)物中找到直系同源物;但有研究表明,魚類與哺乳類的TLRs功能發(fā)生了分化。鯉春病毒血癥病毒(Spring viremia of carp virus,SVCV)是一種彈狀病毒,可引起鯉科魚類及鯰科魚類大規(guī)模暴發(fā)鯉春病毒血癥(Spring viremia of carp,SVC)。SVC傳染快、死亡率高,可造成巨大的經(jīng)濟(jì)損失,已成為鯉科魚類養(yǎng)殖的最大威脅。鯉科魚類是我國淡水養(yǎng)殖的主要品種,產(chǎn)量占淡水養(yǎng)殖的20%以上;其中鯉魚正是SVCV主要的、最易感的宿主,但目前關(guān)于鯉魚抗病毒(SVCV)固有免疫應(yīng)答的知識(shí)非常有限。因此,本論文以鯉魚為研究對(duì)象,對(duì)與病毒先天性識(shí)別有關(guān)的TLRs及其下游信號(hào)通路中的干擾素相關(guān)免疫因子展開研究;并在建立鯉魚-SVCV感染模型的基礎(chǔ)上,從分子角度研究SVCV引起的宿主TLRs、非特異性免疫相關(guān)基因和炎癥相關(guān)基因表達(dá)量的變化,了解鯉魚抗SVCV免疫應(yīng)答的分子機(jī)制,為SVC的免疫防治提供理論指導(dǎo)。第一部分鯉魚抗病毒相關(guān)TLRs功能的研究首先應(yīng)用RT-PCR技術(shù)擴(kuò)增獲得鯉魚TLR2、TLR3、TLR7、TLR9和TLR22基因(Cc TLRs),并將它們克隆入真核表達(dá)載體p EGFP-N1構(gòu)建過表達(dá)質(zhì)粒;然后將過表達(dá)質(zhì)粒p EGFP-N1-Cc TLRs和空載體質(zhì)粒p EGFP-N1同時(shí)轉(zhuǎn)染鯉魚上皮瘤(Epithelioma papulosum cyprinid,EPC)細(xì)胞,采用實(shí)時(shí)熒光定量PCR檢測轉(zhuǎn)染p EGFP-N1-Cc TLR2、p EGFP-N1-Cc TLR9和p EGFP-N1后0 h、6 h、12 h、24 h、48 h和72 h細(xì)胞內(nèi)IRF3和IRF7以及干擾素刺激基因(IFN-stimulated genes,ISGs)ISG15、Mx1、PKR和Viperin(Vig1)的m RNA轉(zhuǎn)錄水平,以及轉(zhuǎn)染p EGFP-N1-Cc TLR3和p EGFP-N1-Cc TLR7后0 h、6 h、12 h、24 h、48 h和72 h細(xì)胞內(nèi)IRF3和IRF7的m RNA轉(zhuǎn)錄水平;并通過SVCV感染實(shí)驗(yàn)探索了TLR2在EPC細(xì)胞中過表達(dá)激發(fā)的抗病毒效應(yīng)。結(jié)果顯示:在EPC細(xì)胞中轉(zhuǎn)染p EGFP-N1-Cc TLR2后IRF3、IRF7、ISG15、Mx1、PKR和Viperin的m RNA轉(zhuǎn)錄水平均出現(xiàn)明顯上調(diào),在轉(zhuǎn)染后48 h,其轉(zhuǎn)錄水平分別相當(dāng)于轉(zhuǎn)染前的6.3倍、16.5倍、15.0倍、13.0倍、7.6倍和92.4倍,差異極顯著(P0.01);與轉(zhuǎn)染p EGFP-N1相比,轉(zhuǎn)染p EGFP-N1-Cc TLR2的EPC細(xì)胞IRF3、IRF7、ISG15、Mx1、PKR和Viperin的m RNA轉(zhuǎn)錄水平在轉(zhuǎn)染后48 h和72 h分別上調(diào)2倍和2.2倍、1.5倍和2.4倍、3.9倍和4.7倍、3.4倍和6.7倍、5.4倍和3.7倍、6.6倍和15.6倍,差異極顯著(P0.01);轉(zhuǎn)染p EGFP-N1組在感染SVCV后6 h、12 h、24 h、48 h和72 h的病毒量分別是轉(zhuǎn)染p EGFP-N1-Cc TLR2組的1.1倍、2.4倍、3.8倍、11.7倍和11.4倍,差異極顯著(P0.01)。因此,鯉魚TLR2在EPC細(xì)胞中過表達(dá)可上調(diào)IRF3、IRF7、ISG15、Mx1、PKR和Viperin基因的m RNA轉(zhuǎn)錄水平,以及抑制SVCV的增殖;提示魚類TLR2可通過激活I(lǐng)RF3或/和IRF7信號(hào)通路誘導(dǎo)I-IFN的產(chǎn)生,進(jìn)而誘導(dǎo)ISG15、Mx1、PKR和Viperin等干擾素刺激基因表達(dá)抗病毒蛋白發(fā)揮抗病毒免疫效應(yīng)。在EPC細(xì)胞中轉(zhuǎn)染p EGFP-N1-Cc TLR9后IRF3、IRF7、ISG15、Mx1、PKR和Viperin的m RNA轉(zhuǎn)錄水平均出現(xiàn)明顯上調(diào),在轉(zhuǎn)染后72 h,其轉(zhuǎn)錄水平分別相當(dāng)于轉(zhuǎn)染前的6.9倍、39.5倍、65.7倍、5.0倍、5.6倍和156.2倍,差異極顯著(P0.01);與轉(zhuǎn)染p EGFP-N1相比,轉(zhuǎn)染p EGFP-N1-Cc TLR9的EPC細(xì)胞IRF3、IRF7、ISG15、Mx1、PKR和Viperin的m RNA轉(zhuǎn)錄水平在轉(zhuǎn)染后48 h和72 h分別上調(diào)1.3倍和2.5倍、2.8倍和3.1倍、5.9倍和10.3倍、3.0倍和1.9倍、2.3倍和2.6倍、6.5倍和11.9倍,差異極顯著(P0.01)。研究表明,鯉魚TLR9在EPC細(xì)胞中過表達(dá)可上調(diào)IRF3、IRF7、ISG15、Mx1、PKR和Viperin基因的m RNA轉(zhuǎn)錄水平,其中Viperin、ISG15和IRF7上調(diào)較明顯,PKR上調(diào)幅度最小;提示魚類TLR9具有哺乳動(dòng)物TLR9相似的功能,能通過激活I(lǐng)RF7信號(hào)通路誘導(dǎo)I-IFN的產(chǎn)生,進(jìn)而誘導(dǎo)ISG15、Mx1和Viperin等干擾素刺激基因表達(dá)抗病毒蛋白發(fā)揮抗病毒免疫效應(yīng)。在EPC細(xì)胞中分別轉(zhuǎn)染p EGFP-N1-Cc TLR3和p EGFP-N1-Cc TLR7,IRF3和IRF7的m RNA轉(zhuǎn)錄水平與轉(zhuǎn)染前相比雖有上調(diào),但與轉(zhuǎn)染p EGFP-N1相比,差異不顯著(P?0.05),說明鯉魚TLR3/TLR7在EPC細(xì)胞中過表達(dá)對(duì)IRF3和IRF7基因的m RNA轉(zhuǎn)錄沒有明顯影響。此外,在本研究中沒有克隆到鯉魚TLR22。第二部分鯉魚免疫相關(guān)基因?qū)VCV感染的應(yīng)激表達(dá)機(jī)制研究通過對(duì)鯉魚注射低于致死劑量的SVCV(實(shí)現(xiàn)了較低的死亡率),研究每條魚Toll樣受體通路中從模式識(shí)別受體到抗病毒基因的各免疫相關(guān)基因應(yīng)激表達(dá)情況。這包括(1)Toll樣受體:TLR2、TLR3和TLR7;(2)干擾素調(diào)節(jié)因子:IRF3和IRF7;(3)干擾素基因:IFNa1、IFNa2和IFNa1S;(4)干擾素刺激基因:ISG15、Mx1、Vig1、PKR和ADAR;(5)促炎性細(xì)胞因子:IL1β、IL6和IL10。結(jié)果顯示:SVCV在3 hpi就開始在宿主細(xì)胞中復(fù)制,6 hpi病毒數(shù)量開始激增,至3 dpi達(dá)到最大值;在5 dpi,病毒復(fù)制開始減少,但10 dpi病毒載量依然處在一個(gè)較高水平。感染后同一時(shí)間點(diǎn)不同樣本體內(nèi)病毒量變化很大,且發(fā)現(xiàn)多數(shù)樣本組有一個(gè)病毒復(fù)制水平高的樣本(33%),這個(gè)比例與死亡率相近,由此推測攜帶病毒多的魚在感染后期會(huì)死亡。鯉魚頭腎中TLRs及其信號(hào)通路中免疫相關(guān)基因表達(dá)量的變化如下:(1)TLR2、TLR3和TLR7的轉(zhuǎn)錄水平在3 hpi就開始出現(xiàn)上調(diào),且分別在3 dpi、12 hpi和1 dpi達(dá)到高峰;隨后開始下降,至10 dpi,3個(gè)TLRs的轉(zhuǎn)錄仍處在一個(gè)低水平的上調(diào)。(2)IRF3和IRF7的轉(zhuǎn)錄水平在3 hpi就開始出現(xiàn)上調(diào),且分別在1 dpi和3 dpi達(dá)到高峰;隨后,IRF3和IRF7的轉(zhuǎn)錄水平開始逐漸下降,但一直處于上調(diào)狀態(tài);且IRF7的上調(diào)幅度比IRF3明顯,至10 dpi,仍處于較高的轉(zhuǎn)錄水平。(3)IFNa1和IFNa2的轉(zhuǎn)錄水平在12 hpi出現(xiàn)明顯上調(diào),并在3 dpi達(dá)到高峰,但I(xiàn)FNa1S的轉(zhuǎn)錄水平一直很低;在5 dpi和7 dpi時(shí),IFNa1和IFNa1S的轉(zhuǎn)錄水平與對(duì)照組相似,而IFNa2一直處在一個(gè)較高水平的上調(diào)。(4)ADAR、ISG15、Mx1、PKR和Vig1的轉(zhuǎn)錄水平在12 hpi開始出現(xiàn)明顯上調(diào);其中ADAR、ISG15和PKR在1 dpi上調(diào)達(dá)到高峰,3 dpi后開始逐漸下降,但在整個(gè)實(shí)驗(yàn)期間仍保持上調(diào)狀態(tài);Mx1和Vig1在1 dpi和3 dpi高水平上調(diào)表達(dá)并達(dá)到高峰,在5 dpi減少,但表達(dá)水平仍相對(duì)較高。(5)IL1β、IL6和IL10的轉(zhuǎn)錄水平在3 hpi和6 hpi時(shí)出現(xiàn)下調(diào),但在12 hpi和1 dpi時(shí)上調(diào)至較高水平;隨后IL6和IL10出現(xiàn)下調(diào),而IL1β則一直保持上調(diào)狀態(tài)。從上述研究結(jié)果看,各免疫相關(guān)基因可受病毒刺激而上調(diào)表達(dá),表達(dá)量基本與體內(nèi)病毒量成正比。綜上,鯉魚感染SVCV后,病毒在很短的時(shí)間內(nèi)開始在宿主細(xì)胞中復(fù)制,并以指數(shù)速率增加;病毒感染早期,TLR2、TLR3和TLR7迅速上升到一個(gè)比較高的水平,啟動(dòng)免疫刺激信號(hào),刺激IRFs、I-IFNs和ILs的表達(dá);在病毒感染的中后期,ADAR、ISG15、Mx1、PKR和Vig1等干擾素刺激基因大量表達(dá)發(fā)揮抗病毒免疫效應(yīng)。因此,SVCV(彈狀病毒)可以刺激機(jī)體上調(diào)先天性免疫因子的表達(dá)以抵抗病毒,且主要以I-IFNs介導(dǎo)的抗病毒免疫應(yīng)答為主,即TLR2、TLR3和TLR7識(shí)別病毒后可能能夠通過IRFs/I-IFNs/ISGs信號(hào)途徑激活抗病毒反應(yīng)。
[Abstract]:Toll like receptors (Toll-like receptors TLRs) is a viral pathogen associated molecular pattern recognition (Pattern associated molecular patterns, PAMPs) are pattern recognition receptors (Pattern recognition, receptors, PRRs), the identification of PAMPs virus, D88 (Myeloid differentiation My based on factor 88, My D88) or TRIF (TIR domain-containing adaptor-inducing interferon- beta, TRIF) signal transduction pathway induced by nuclear factor kappa B (factor- K Nuclear B NF- K B) and interferon regulatory factor (Interferon regulatory, factors, IRFs) expression, and initiate innate and adaptive immunity against invading viruses. So far in the teleost fish have been identified in 20 kinds of TLRs, TLRs can find the most fish orthologues in mammals; but research has shown that the differentiation of fish and mammalian TLRs. Spring viraemia of carp (Spring viremia of car P virus, SVCV) is a rhabdovirus, can cause cyprinid fish and grouper large-scale outbreaks of spring viremia (Spring viremia of carp, SVC).SVC infection quickly, the mortality rate is high, can cause huge economic losses, has become the biggest threat of cyprinid fish breeding. Carp is the main freshwater aquaculture species in China, freshwater aquaculture production accounts for more than 20%; the main carp is SVCV, the most susceptible hosts, but on the common carp (SVCV) antiviral innate immune response knowledge is very limited. Therefore, the carp as the research object, to study the virus and identification of congenital interferon immune factors related to TLRs and its downstream signaling pathway; and based on common carp -SVCV infection model, SVCV research from the perspective of molecular host TLRs genes expression of genes related to nonspecific immunity and inflammation Change, understand the molecular mechanism of carp anti SVCV immune response, to provide theoretical guidance for the prevention of immune SVC. The first part of the study of carp antiviral related TLRs function is first applied to RT-PCR was amplified from TLR2 TLR3, TLR7, carp, TLR9 and TLR22 genes (Cc TLRs), and they are cloned into the eukaryotic expression vector of P EGFP-N1 construction of expression plasmid; then overexpression plasmid P EGFP-N1-Cc TLRs and empty plasmid P EGFP-N1 and transfection of carp (Epithelioma papulosum cyprinid, epithelial tumor cells, EPC) detected by real-time fluorescent quantitative PCR EGFP-N1-Cc TLR2 P EGFP-N1-Cc was transfected into P, TLR9 and P EGFP-N1 after 0 h, 6 h, 12 h, 24 h, 48 h and H 72 cells IRF3 and IRF7 and interferon stimulated genes (IFN-stimulated genes, ISGs) ISG15, Mx1, PKR and Viperin (Vig1) m RNA and P EGFP-N1-Cc transcription, transfection of TLR3 and P EGFP-N1-Cc TLR7 0 h, 6 h, 12 h, 24 h,48 h鍜，

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