本文選題：流感病毒　切入點(diǎn)：miRNA　出處：《吉林大學(xué)》2015年碩士論文

【摘要】：A型流感病毒（influenza A virus，IAV）是單股負(fù)鏈RNA病毒，其首先侵染的是宿主的呼吸系統(tǒng)，臨床表現(xiàn)從輕微的上呼吸道感染到嚴(yán)重的肺炎。MicroRNA(miRNA)屬于非編碼RNA家族，在轉(zhuǎn)錄后水平調(diào)控靶基因表達(dá)。目前在各種生物中已經(jīng)確定了成千上萬種miRNA，其參與了細(xì)胞分化、增殖、凋亡和病原感染等諸多生物學(xué)進(jìn)程的調(diào)控。人的miR-29家族包括miR-29a、miR-29b及miR-29c。最近的研究顯示，miR-29能夠調(diào)節(jié)機(jī)體的天然免疫和適應(yīng)性免疫，但其在IAV感染過程中對(duì)于宿主免疫反應(yīng)的作用機(jī)制尚不明確。 我們前期miRNA組學(xué)的結(jié)果表明，當(dāng)機(jī)體感染IAV后，miR-29家族的表達(dá)顯著上調(diào)，，尤其是miR-29c。圍繞miR-29c我們進(jìn)行了如下的研究。第一，首先分析了先前的關(guān)于IAV不同時(shí)相感染A549細(xì)胞的miRNA表達(dá)譜數(shù)據(jù)，并應(yīng)用Real-time PCR對(duì)表達(dá)差異顯著的miR-29c進(jìn)行了驗(yàn)證。預(yù)測(cè)得到了miR-29c的潛在靶基因集合，并對(duì)此進(jìn)行生物信息學(xué)數(shù)據(jù)挖掘，其中由于A20是一個(gè)重要的機(jī)體炎癥和抗病毒信號(hào)通路的調(diào)節(jié)蛋白，對(duì)于維持機(jī)體的免疫穩(wěn)態(tài)具有重要作用，據(jù)此篩選出miR-29c/A20互作對(duì)作為研究的切入點(diǎn)；第二，利用雙熒光素酶報(bào)告基因技術(shù)、Real-time PCR以及Western Blotting實(shí)驗(yàn)對(duì)miR-29c與A20之間的相互作用機(jī)制進(jìn)行了確證，并證明IAV能夠有效增加A20mRNA的穩(wěn)定性，且通過過表達(dá)和表達(dá)抑制的系列實(shí)驗(yàn)證明IAV通過誘導(dǎo)miR-29c的表達(dá)促進(jìn)了細(xì)胞A20的表達(dá)；第三，在功能學(xué)實(shí)驗(yàn)部分，先利用熒光素酶報(bào)告基因和WesternBlotting技術(shù)證明miR-29c/A20能有效抑制NF-κ B信號(hào)通路的激活。我們通過Real-time PCR方法對(duì)miR-29c/A20互作對(duì)在IAV感染A549細(xì)胞內(nèi)TNF-α、IFN-β、 IL-1β、 IL-6、以及IL-8等細(xì)胞因子的mRNA表達(dá)變化進(jìn)行分析，證明miR-29c能通過上調(diào)A20的表達(dá)來抑制IAV誘導(dǎo)的NF-κ B下游抗病毒和促炎細(xì)胞因子的表達(dá)。 綜上所述，IAV的感染可以通過上調(diào)miR-29c進(jìn)而增加A20的表達(dá)來抑制宿主天然抗病毒免疫。
[Abstract]:Influenza A virus type A virus is a single negative strand RNA virus, which first infects the host's respiratory system. Its clinical manifestations range from mild upper respiratory tract infection to severe pneumonia. MicroRNAs belong to a non-coding RNA family and regulate the expression of target genes at post-transcriptional level.At present, thousands of miRNAs have been identified in various organisms, which are involved in many biological processes such as cell differentiation, proliferation, apoptosis and pathogenic infection.The human miR-29 family includes miR-29aanmiR-29b and miR-29c.Recent studies have shown that miR-29 can regulate innate and adaptive immunity, but its mechanism of host immune response in the process of IAV infection is unclear.The results of our previous miRNA studies showed that the expression of miR-29 was significantly up-regulated when the body was infected with IAV, especially miR-29c.We have done the following research around miR-29c.Firstly, the previous data of miRNA expression profile of A549 cells infected with IAV at different time were analyzed, and Real-time PCR was used to verify the expression of miR-29c.The potential target gene sets of miR-29c were predicted, and the bioinformatics data were mined. Because A20 is an important regulatory protein of inflammation and antiviral signaling pathway, it plays an important role in maintaining the immune homeostasis of the body.The interaction between miR-29c and A20 was confirmed by using double luciferase reporter gene technique: Real-time PCR and Western Blotting experiments, and it was proved that IAV could effectively increase the stability of A20mRNA.It was proved by a series of experiments of overexpression and inhibition that IAV promoted the expression of A20 by inducing the expression of miR-29c.Luciferase reporter gene and WesternBlotting technique were used to prove that miR-29c/A20 could effectively inhibit the activation of NF- 魏 B signaling pathway.We analyzed the mRNA expression of TNF- 偽 -IFN- 尾, IL-1 尾, IL-6, IL-8 and other cytokines in A549 cells infected with IAV by Real-time PCR.It is suggested that miR-29c can inhibit the expression of antiviral and pro-inflammatory cytokines induced by IAV by up-regulating A20 expression.In conclusion, miR-29c infection can inhibit host natural anti-viral immunity by upregulating miR-29c and increasing A20 expression.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.65

【共引文獻(xiàn)】

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7 王彥s

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