【摘要】：細(xì)胞先天性免疫的第一道防線是那些能識別病源菌保守成分的受體。研究發(fā)現(xiàn)草魚感染嗜水氣單胞菌引發(fā)細(xì)菌性敗血癥。在感染轉(zhuǎn)錄組數(shù)據(jù)中發(fā)現(xiàn)TLR受體(Toll-like receptor)基因是差異表達(dá)基因,這說明TLR基因家族參與機體抵御外源性致病菌的入侵。另外組織表達(dá)譜結(jié)果顯示,大部分TLR基因在免疫組織中高表達(dá),比如皮膚、脾臟、肝和鰓等。從轉(zhuǎn)錄組水平和組織表達(dá)模式都說明TLR參與與機體免疫反應(yīng),因此對TLR基因全面、系統(tǒng)化的研究尤為重要。本文基于草魚基因組、嗜水氣單胞菌感染脾臟轉(zhuǎn)錄組、以及嗜水氣單胞菌抗病組和易感組轉(zhuǎn)錄組數(shù)據(jù)深度剖析TLR基因具體信息,對已完成TLR18,TLR20和TLR21cDNA克隆的基礎(chǔ)上進行功能性探究。主要結(jié)果如下:草魚基因組有20種TLR基因,分屬于6個亞家族,基因組中尚未發(fā)現(xiàn)TLR14,TLR16,TLR19和TLR26基因,卻發(fā)現(xiàn)草魚特有的TLR27基因。系統(tǒng)發(fā)育和共線性分析顯示TLR11亞家族是最大的分支,TLR11家族基因共線性分析結(jié)果和洞穴魚、斑馬魚、河豚和羅非魚相似。TLR基因組序列分析,大部分TLR基因沒有內(nèi)含子,屬于一個亞分支上的TLR基因一般有相似的內(nèi)含子/外顯子結(jié)構(gòu)和數(shù)目。正常組織表達(dá)譜表明TLR5b,TLR5a,TLR1和TLR25表達(dá)量相對比較高,但總體TLR基因RPKM比較低,約為144.73。草魚嗜水氣單胞菌易感和抗病轉(zhuǎn)錄組中有9個TLR基因差異表達(dá);脾臟轉(zhuǎn)錄組中6個TLR基因上調(diào),上調(diào)或抑制的程度為2到12.9倍。其中TLR20.2上調(diào)最為顯著,在感染48h時,該基因上調(diào)了12.9倍。體外gctlr18誘導(dǎo)表達(dá),利用鞭毛蛋白,LPS和poly(I:C)分別刺激CIK細(xì)胞系,發(fā)現(xiàn)gctlr18的表達(dá)量均呈現(xiàn)先上升后下降的趨勢,分別在24h,48h和12h表達(dá)量到達(dá)峰值,誘導(dǎo)倍數(shù)分別是9.78倍,8.87倍和8.92倍。過表達(dá)gctlr18基因?qū)l-8,inf-1和tnf-α有調(diào)控作用,分別上升1.2倍,10.15倍和3.61倍,其中對il-8沒有顯著誘導(dǎo)性。過表達(dá)gctlr18能激活NF-κB熒光報告系統(tǒng),以及抵御細(xì)菌入侵的作用,細(xì)菌侵染數(shù)比對照組少83.4%。因此,gctlr18可能識別鞭毛蛋白,LPS和poly(I:C)致病成分,gctlr18過表達(dá)能誘導(dǎo)炎癥因子分泌,可能是通過NF-κB信號通路引發(fā)炎癥以清除外源性病原菌。將TLR20 cDNA序列根據(jù)草魚基因組數(shù)據(jù)庫比對得到該基因?qū)儆趃ctlr20.2,感染嗜水氣單胞菌草魚脾臟轉(zhuǎn)錄組中屬于差異表達(dá)基因。體外刺激實驗表明,鞭毛蛋白,LPS和poly(I:C)能刺激誘導(dǎo)gctlr20.2的表達(dá),鞭毛蛋白和poly(I:C)在24h達(dá)到峰值3.35倍和2.8倍,LPS刺激后6h時表達(dá)量最高2.07倍。過表達(dá)和干擾該基因?qū)ο掠蚊庖呦嚓P(guān)基因有調(diào)控作用,gctlr20.2過表達(dá)誘導(dǎo)il1β,il8和tnf-α轉(zhuǎn)錄增加,il1β上調(diào)1.46倍,il8上調(diào)1.45倍,tnf-α上調(diào)1.91倍。siRNA-tlr20.2對gctlr20.2mRNA有65.7%的抑制效率,并且下調(diào)ifn和il8基因的轉(zhuǎn)錄,分別為0.77倍和0.14倍。因此,gctlr20.2也能識別鞭毛蛋白,LPS和poly(I:C)致病成分,gctlr20.2過表達(dá)激活NF-κB信號通路引發(fā)炎癥反應(yīng)。下調(diào)gctlr20.2能夠減少炎癥因子分泌,有助于機體恢復(fù)免疫平衡。鞭毛蛋白和LPS體外刺激gctlr21表達(dá),同時過表達(dá)和干擾該基因?qū)ο掠蚊庖呦嚓P(guān)基因有調(diào)控作用。LPS感染6h時,表達(dá)量上升到2.45倍,到24h到達(dá)峰值8.63倍,隨后緩慢下降。FLA-ST感染6h時,轉(zhuǎn)錄水平上升至2.7倍,24h時出現(xiàn)峰值5.61倍,48h后下降到2.22倍。Gctlr21過表達(dá)誘導(dǎo)il1β和ifn表達(dá)顯著性上調(diào),分別為2.12倍和1.66倍,并且激活NF-κB熒光報告系統(tǒng)。Sitlr21-2對gctlr21表達(dá)抑制效果為0.48倍,并顯著性抑制下游免疫因子il1β,il8和tnf-α的表達(dá),對ifn抑制效果不明顯。篩選靶調(diào)控microRNA,發(fā)現(xiàn)Ci-miR-3,let-7i和Ci-miR-14對gctlr21基因轉(zhuǎn)錄有抑制作用。過表達(dá)microRNA對免疫相關(guān)基因也有調(diào)控作用。因此,gctlr21基因可能也參與鞭毛蛋白和LPS的識別過程,推測gctlr21過表達(dá)能激活NF-κB調(diào)控炎癥因子的轉(zhuǎn)錄。RNA干擾的效果與Ci-mi R-3,let-7i和Ci-miR-14也可以靶調(diào)控gctlr21的表達(dá)效果類似,推測gctlr21轉(zhuǎn)錄下調(diào)能減弱炎癥反應(yīng)避免機體過度損傷。
[Abstract]:The first line of defense against cellular innate immunity is those receptors that recognize the conserved components of pathogenic bacteria. Studies have found that grass carp infected with Aeromonas hydrophila cause bacterial sepsis. In addition, the results of tissue expression profiles showed that most TLR genes were highly expressed in immune tissues, such as skin, spleen, liver and gill. The transcriptome level and tissue expression patterns showed that TLR was involved in the immune response of the organism. Therefore, it is particularly important to study the TLR gene comprehensively and systematically. The transcriptome data of Aeromonas spp. infected spleen, Aeromonas hydrophila resistant and susceptible groups were analyzed in depth to explore the function of TLR gene based on the cloning of TLR18, TLR2 0 and TLR21. TLR14, TLR16, TLR19, and TLR26 genes were found to be unique to grass carp. Phylogenetic and collinear analyses showed that the TLR11 subfamily was the largest branch. The TLR11 family was similar to cave fish, zebrafish, puffer fish and tilapia in gene analysis. Normal tissue expression profiles showed relatively high levels of TLR5b, TLR5a, TLR1 and TLR25, but the overall TLR gene RPKM was relatively low, about 144.73. Nine TLR genes were differentially expressed in the susceptible and resistant transcripts of Aeromonas hydrophila. TLR genes were up-regulated and up-regulated by 2 to 12.9 times. TLR20.2 was up-regulated most significantly, and up-regulated by 12.9 times at 48h after infection. The expression of gctlr18 was induced by flagellin, LPS and poly (I:C) in vitro. The expression of gctlr18 increased first and then decreased, respectively, at 24h, 48h and 1h after infection. Overexpression of gctlr18 gene could regulate il-8, inf-1 and TNF-a by 1.2 times, 10.15 times and 3.61 times, respectively. overexpression of gctlr18 could activate the NF-kappa B fluorescence reporting system and resist bacterial invasion. Therefore, gctlr18 may recognize flagellin, LPS and poly (I:C) pathogenic components. Overexpression of gctlr18 can induce the secretion of inflammatory factors, possibly through the NF-kappa B signaling pathway to cause inflammation to eliminate exogenous pathogens. Flagellin, LPS and poly (I: C) stimulated the expression of gctlr20.2 in vitro. Flagellin and poly (I: C) peaked at 3.35 and 2.8 times at 24 h, and the highest expression was 2.07 times at 6 h after LPS stimulation. Overexpression and interference with the gene were immune to the downstream. Overexpression of gctlr20.2 induced an increase in the transcription of IL-1 beta, IL-8 and tnf-a, up-regulation of IL-1 beta by 1.46 times, up-regulation of IL-8 by 1.45 times and up-regulation of TNF-a by 1.91 times. siRNA-tlr20.2 inhibited the expression of gctlr20.2 mRNA by 65.7%, and down-regulated the transcription of IFN and IL-8 genes by 0.77 and 0.14 times, respectively. LPS and poly(I:C) pathogenic components, gctlr20.2 overexpression activates NF-kappa B signaling pathway to trigger inflammation. Downregulation of gctlr20.2 can reduce the secretion of inflammatory factors and help to restore immune balance. Flagellin and LPS stimulate the expression of gctlr21 in vitro, and over-expression and interference of the gene may regulate the downstream immune-related genes in LPS infection. At 6 h, the expression increased to 2.45 times, reached a peak value of 8.63 times at 24 h, and then decreased slowly. At 6 h, the transcriptional level of FLA-ST increased to 2.7 times, reached a peak value of 5.61 times at 24 h, and decreased to 2.22 times at 48 h. Overexpression of Gctlr21 induced significant up-regulation of IL-1 beta and IFN expression, 2.12 times and 1.66 times respectively, and activated the NF-kappa B fluorescence reporting system. 2 inhibited the expression of gctlr21 by 0.48 times, and significantly inhibited the expression of downstream immune factors IL-1 beta, IL-8 and tnf-a, but did not inhibit the expression of ifn. The Target-regulated microRNA screening showed that Ci-Mi-3, let-7i and CI-Mi-14 inhibited the transcription of gctlr21 gene. Overexpression of microRNA also regulated the expression of immune-related genes. Genes may also be involved in the recognition of flagellin and LPS, suggesting that the overexpression of gctlr21 can activate NF-kappa B to regulate the transcription of inflammatory factors. RNA interference is similar to that of CI-mi R-3, let-7i and CI-microRNA-14 in targeting the expression of gctlr21, suggesting that the down-regulation of gctlr21 transcription can attenuate inflammation and avoid excessive injury.
【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S943

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