【摘要】：T-2毒素由鐮刀菌產(chǎn)生,是單端孢霉烯族類(lèi)毒素中毒性最強(qiáng)的毒素。通過(guò)污染飼料,T-2毒素對(duì)動(dòng)物和動(dòng)物性食品消費(fèi)者的健康造成嚴(yán)重危害。研究表明,T-2毒素主要損傷動(dòng)物機(jī)體的免疫系統(tǒng)。該類(lèi)毒素能使機(jī)體在低劑量的暴露下引起免疫刺激,但在高劑量的暴露下引起免疫抑制,導(dǎo)致免疫應(yīng)答水平被嚴(yán)重干擾,機(jī)體免疫功能紊亂。這類(lèi)毒素細(xì)胞毒性的發(fā)揮主要是作用于核糖體,一方面可以與肽酰轉(zhuǎn)移酶催化位點(diǎn)結(jié)合來(lái)阻礙生物大分子如蛋白質(zhì)等的生成,另一方面通過(guò)核糖體應(yīng)激反應(yīng)快速激活絲裂原活化蛋白激酶(MAPK)通路,使相關(guān)免疫應(yīng)答的下游因子的表達(dá)在轉(zhuǎn)錄和轉(zhuǎn)錄后水平受到調(diào)控。其中被誘導(dǎo)的促炎性細(xì)胞因子的基因表達(dá)上調(diào)是促使T-2毒素發(fā)揮各種毒性效應(yīng)的主要介質(zhì)。MicroRNA-155作為一個(gè)典型的多功能的microRNA,同眾多microRNA一樣,也是通過(guò)與靶基因的3’UTR結(jié)合,抑制基因的表達(dá)。MicroRNA-155抑制超過(guò)60種靶基因,可以通過(guò)靶向不同的基因而發(fā)揮不同的作用。研究表明它不僅在炎癥反應(yīng)中可以被快速激活,而且受到刺激差異性表達(dá)的microRNA-155在免疫調(diào)節(jié)反應(yīng)中起著非常關(guān)鍵的作用。但是關(guān)于microRNA-155是否在T-2毒素介導(dǎo)的炎癥反應(yīng)中發(fā)揮作用仍未見(jiàn)報(bào)道。本研究以RAW264.7細(xì)胞為模型,初步探究了microRNA-155在T-2毒素誘導(dǎo)下對(duì)炎癥因子的調(diào)控作用機(jī)制。本研究首先用qRT-PCR的方法檢測(cè)了T-2毒素處理下microRNA-155是否差異性表達(dá),結(jié)果顯示,14nM的T-2毒素處理RAW264.7細(xì)胞1h后microRNA-155上調(diào),2h時(shí)上調(diào)最明顯(p0.01),后續(xù)雖有下調(diào),但與空白組相比仍明顯上調(diào)。這種上調(diào)也出現(xiàn)在不同濃度(7nM、14nM、28nM)的T-2毒素處理RAW264.7細(xì)胞12h后。其中7nM時(shí),microRNA-155上調(diào)最顯著(p0.01)。說(shuō)明T-2毒素可以促進(jìn)RAW264.7細(xì)胞中microRNA-155表達(dá)。其次利用microRNA-155 mimic和抑制劑(inhibitor)分別過(guò)表達(dá)和抑制細(xì)胞中microRNA-155的表達(dá)水平,T-2毒素處理后發(fā)現(xiàn),IL-6(p0.01)、IL-1β(p0.05)和TNF-α(p0.01)的表達(dá)水平隨microRNA-155的上調(diào)而顯著上升,隨microRNA-155下調(diào)而下降(p0.05),說(shuō)明microRNA-155參與并促進(jìn)T-2毒素誘導(dǎo)的促炎癥因子的表達(dá)。SOCS1是JAK/STAT通路的負(fù)性調(diào)節(jié)因子,被驗(yàn)證是microRNA-155的靶標(biāo)。microRNA-155在多種情況下都可以通過(guò)直接抑制SOCS1發(fā)揮促炎的作用。但是在T-2毒素的誘導(dǎo)下這種調(diào)節(jié)作用是否存在卻未可知。于是分別通過(guò)mimic和inhibitor上調(diào)和下調(diào)了microRNA-155的表達(dá),發(fā)現(xiàn)SOCS1的表達(dá)在microRNA-155上調(diào)時(shí)顯著下降(p0.01),在其下調(diào)時(shí)顯著上升(p0.01)。說(shuō)明在T-2毒素的誘導(dǎo)下,microRNA-155通過(guò)靶向抑制SOCS1發(fā)揮了促炎的作用。每一個(gè)microRNA都有多個(gè)靶基因,通過(guò)靶向不同的基因發(fā)揮不同的作用。通過(guò)生物信息學(xué)方法確定了兩個(gè)與炎癥相關(guān)的microRNA-155的靶標(biāo)rheb和atg3。Rheb是Ras同源基因,廣泛存在于多種生物中,是一個(gè)高度保守的GTP結(jié)合蛋白,并且是mTOR的上游關(guān)鍵調(diào)控因子,參與調(diào)節(jié)機(jī)體的如細(xì)胞生長(zhǎng)、分化、氧化應(yīng)激等眾多生物學(xué)功能。Atg3是一種類(lèi)似于泛素化過(guò)程中E2酶的一種催化蛋白,作為自噬家族中的一員,連接LC3和磷脂酰乙醇胺,通過(guò)脂化蛋白質(zhì),降解長(zhǎng)壽命蛋白質(zhì)和已損壞或不必要的細(xì)胞器,在囊泡或溶酶體系統(tǒng)進(jìn)行抗原提呈、抗感染、細(xì)胞分化、細(xì)胞發(fā)育等生物學(xué)過(guò)程中發(fā)揮重要作用。本實(shí)驗(yàn)分別通過(guò)mimic和inhibitor上調(diào)和下調(diào)了microRNA-155的表達(dá),發(fā)現(xiàn)rheb和atg3的mRNA水平和蛋白水平均在microRNA-155上調(diào)時(shí)顯著下降,在其下調(diào)時(shí)顯著上升。分別構(gòu)建了含rheb和atg3 3’UTR結(jié)合位點(diǎn)序列的pGL3野生質(zhì)粒和突變質(zhì)粒,將microRNA-155 mimic和NC分別與這兩個(gè)質(zhì)粒組合轉(zhuǎn)染進(jìn)RAW264.7細(xì)胞內(nèi)進(jìn)行了雙熒光素酶報(bào)告基因?qū)嶒?yàn)。結(jié)果顯示:microRNA-155對(duì)p GL3-3’UTR野生型的熒光素酶活性的表達(dá)有極顯著的抑制作用(p0.01),熒光素酶的活性均下調(diào)60%左右,說(shuō)明了microRNA-155分別通過(guò)與rheb和atg3的3’UTR相互作用,抑制了rheb和atg3的表達(dá),rheb和atg3是microRNA-155的直接靶標(biāo)。通過(guò)構(gòu)建pcDNA-rheb顯著上調(diào)細(xì)胞內(nèi)rheb表達(dá)水平(p0.01)后發(fā)現(xiàn),IL-6、IL-1β和TNF-α的表達(dá)水平顯著上升,說(shuō)明rheb在T-2毒素誘導(dǎo)下可以促進(jìn)炎癥因子的表達(dá)。通過(guò)siRNA方法干擾細(xì)胞中rheb表達(dá)后,炎癥因子的表達(dá)變化卻不明顯,猜測(cè)可能原因rheb只是T-2毒素誘導(dǎo)的促進(jìn)炎癥因子表達(dá)的其中一種因素,當(dāng)rheb表達(dá)減少時(shí),其他因素或許代償性增加,導(dǎo)致對(duì)炎癥因子的影響并不大。最后得出microRNA-155在T-2毒素的誘導(dǎo)下通過(guò)抑制rheb發(fā)揮抗炎的作用。通過(guò)構(gòu)建pcDNA-atg3顯著上調(diào)細(xì)胞內(nèi)atg3表達(dá)水平(p0.01)后發(fā)現(xiàn),IL-6極顯著下調(diào)(p0.01),IL-1β和TNF-α略微下調(diào),說(shuō)明上調(diào)的atg3可以抑制T-2毒素誘導(dǎo)的促炎癥因子的表達(dá)。細(xì)胞內(nèi)atg3在經(jīng)過(guò)siRNA下調(diào)之后,T-2毒素再處理12h,IL-6(p0.05)、IL-1β(p0.01)和TNF-α(p0.01)明顯上調(diào),說(shuō)明下調(diào)的atg3可以促進(jìn)T-2毒素誘導(dǎo)的促炎癥因子的表達(dá)。由此證明了microRNA-155通過(guò)直接靶向調(diào)節(jié)atg3來(lái)促進(jìn)T-2毒素誘導(dǎo)的炎癥因子的產(chǎn)生。綜上,本文發(fā)現(xiàn)在T-2毒素誘導(dǎo)下,rheb具有促進(jìn)炎癥因子表達(dá)的作用,atg3具有抑制炎癥因子表達(dá)的作用,并且是microRNA-155的新靶標(biāo)。MicroRNA-155在T-2毒素的誘導(dǎo)下發(fā)揮了雙重的作用。它一方面通過(guò)靶向抑制SOCS1和atg3發(fā)揮促炎的作用,另一面通過(guò)靶向抑制rheb發(fā)揮抗炎的作用,防止炎癥的進(jìn)一步過(guò)大。進(jìn)一步完善了microRNA-155的功能研究,有助于深入闡釋T-2毒素的毒性機(jī)制。為T(mén)-2毒素的功能研究、發(fā)展有效的安全評(píng)估和開(kāi)發(fā)出高效的拮抗劑奠定科學(xué)基礎(chǔ)。
[Abstract]:T-2 toxin, produced by Fusarium spp., is the most poisonous toxin of the monotetramethylene toxoid. By contaminating feedstuffs, T-2 toxin causes serious harm to the health of animals and consumers of animal food. Studies have shown that T-2 toxin mainly damages the immune system of animals. This toxin can cause immunity in low doses of exposure. The cytotoxicity of these toxins mainly acts on the ribosome. On the one hand, they can bind to peptidyltransferase catalytic sites to inhibit the production of biological macromolecules such as proteins, on the other hand, they can interfere with the level of immune response and disturb the immune function of the body. Ribosomal stress reacts rapidly to activate the mitogen-activated protein kinase (MAPK) pathway, which regulates the expression of downstream factors associated with immune responses at both transcriptional and post-transcriptional levels. Increased expression of inducible pro-inflammatory cytokines is the main mediator for T-2 toxins to exert various toxic effects. MicroRNA-155 acts as one of the major mediators. MicroRNA-155 inhibits more than 60 target genes and plays different roles by targeting different genes. Studies have shown that it can not only be activated rapidly in inflammation, but also be poorly stimulated. Heterosexual expression of microRNA-155 plays a key role in the immune regulatory response. However, it has not been reported whether microRNA-155 plays a role in T-2 toxin-mediated inflammatory response. The results showed that the expression of microRNA-155 in RAW264.7 cells treated with 14nM T-2 toxin was up-regulated after 1 hour and up-regulated most significantly after 2 hours (p0.01). Although down-regulated, the expression of microRNA-155 was still up-regulated in different concentrations (7nM, 14nM, 28nM). Twelve hours after treatment with T-2 toxin, the expression of microRNA-155 in RAW264.7 cells was up-regulated most significantly at 7 nM (p0.01). This indicated that T-2 toxin could promote the expression of microRNA-155 in RAW264.7 cells. Secondly, microRNA-155 mic and inhibitor were used to overexpress and inhibit the expression of microRNA-155 in RAW264.7 cells. After treatment with T-2 toxin, IL-6 (p0.0) was found. 1), IL-1 beta (p0.05) and TNF-alpha (p0.01) expression levels increased significantly with the increase of microRNA-155, but decreased with the decrease of microRNA-155 (p0.05), indicating that microRNA-155 participated in and promoted the expression of pro-inflammatory factors induced by T-2 toxin. SOCS1 is a negative regulator of JAK/STAT pathway, and has been proved to be a target of microRNA-155. It was found that the expression of microRNA-155 was up-regulated and down-regulated by mimic and inhibitor respectively, and the expression of SOCS1 was down-regulated significantly when the expression of microRNA-155 was up-regulated (p0.01). MicroRNA-155 plays an inflammatory role by targeting SOCS1. Each microRNA has multiple target genes and plays different roles by targeting different genes. Two inflammation-related microRNA-155 targets, rheb and atg3, were identified by bioinformatics. Atg3 is a highly conserved GTP binding protein and a key regulator upstream of mTOR. It is involved in many biological functions such as cell growth, differentiation, oxidative stress and so on. Atg3 is a catalytic protein similar to E2 enzymes during ubiquitination, and is one of the autophagy families. Members, linked to LC3 and phosphatidylethanolamine, degrade long-lived proteins and damaged or unnecessary organelles by lipoylation of proteins, play important roles in the biological processes of antigen presentation, anti-infection, cell differentiation, and cell development in vesicle or lysosome systems. The expression of oRNA-155 showed that both the mRNA and protein levels of rheb and atg3 decreased significantly when microRNA-155 was up-regulated and increased significantly when microRNA-155 was down-regulated. The results showed that microRNA-155 inhibited the expression of luciferase in wild-type P GL3-3'UTR significantly (p0.01). The activity of luciferase was down by about 60%, indicating that microRNA-155 inhibited rheb and ATG 3 by interacting with 3'UTR of rheb and ATG 3, respectively. The expression of rheb and atg3 was the direct target of microRNA-155. After constructing pcDNA-rheb, the expression of rheb was up-regulated significantly (p0.01). The expression of IL-6, IL-1beta and TNF-alpha was up-regulated significantly, indicating that rheb could promote the expression of inflammatory factors induced by T-2 toxin. It is speculated that rheb is only one of the factors that promote the expression of inflammatory factors induced by T-2 toxin. When the expression of rheb decreases, other factors may increase compensatively, resulting in little effect on inflammatory factors. Finally, it is concluded that microRNA-155 plays an anti-inflammatory role by inhibiting rheb induced by T-2 toxin. After constructing pcDNA-atg3, the expression of atg3 was significantly up-regulated (p0.01), IL-6 was significantly down-regulated (p0.01), IL-1 beta and TNF-a were slightly down-regulated, indicating that the up-regulated atg3 could inhibit the expression of pro-inflammatory factors induced by T-2 toxin. In conclusion, rheb can promote the expression of inflammatory factors induced by T-2 toxin, and atg3 can inhibit the expression of inflammatory factors induced by T-2 toxin. MicroRNA-155 plays a dual role in the induction of T-2 toxin. On the one hand, it plays a pro-inflammatory role by targeting the inhibition of SOCS1 and atg3, on the other hand, it plays an anti-inflammatory role by targeting the inhibition of rheb to prevent inflammation further. The functional study of A-155 will help to elucidate the toxic mechanism of T-2 toxin and lay a scientific foundation for the functional study of T-2 toxin, the development of effective safety assessment and the development of effective antagonists.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S859.8

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;MicroRNA signatures in liver diseases[J];World Journal of Gastroenterology;2009年14期

相關(guān)博士學(xué)位論文 前1條

1 吳慶華;T-2毒素的代謝和毒作用信號(hào)轉(zhuǎn)導(dǎo)研究[D];華中農(nóng)業(yè)大學(xué);2014年

相關(guān)碩士學(xué)位論文 前1條

1 柳芹;JAK/STAT信號(hào)通路在DON和T-2毒素對(duì)RAW264.7細(xì)胞毒性作用中的機(jī)制研究[D];華中農(nóng)業(yè)大學(xué);2011年

，

本文編號(hào)：2215077