本文關(guān)鍵詞：白介素-7促進(jìn)狂犬病病毒免疫記憶的作用機(jī)制研究　出處：《華中農(nóng)業(yè)大學(xué)》2017年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 狂犬病病毒 白介素-7 濾泡性T輔助細(xì)胞 生發(fā)中心B細(xì)胞 記憶性B細(xì)胞 漿細(xì)胞 免疫記憶 體液免疫

【摘要】：狂犬病(Rabies)作為最古老的傳染病之一,是由狂犬病病毒(Rabies Virus,RABV)引起的人獸共患中樞神經(jīng)系統(tǒng)傳染病,分布于世界大部分地區(qū)。我國(guó)每年死于狂犬病的人數(shù)居世界第二位及全國(guó)各類(lèi)傳染病死亡人數(shù)的前五位,其中95%以上的病例是因直接或間接接觸患病或帶毒的動(dòng)物引發(fā)的。目前,狂犬病發(fā)病后的臨床治療仍是一個(gè)世界性難題,其致死率高達(dá)100%,尚無(wú)有效的治療手段。數(shù)據(jù)顯示,70%以上犬的免疫覆蓋率可以有效控制人間狂犬病,因此,動(dòng)物的免疫預(yù)防成為遏制人類(lèi)狂犬病最為有效的措施。當(dāng)前用于動(dòng)物狂犬病免疫的疫苗主要為安全的滅活疫苗,但價(jià)格昂貴,并需多次免疫才能獲得良好的長(zhǎng)期保護(hù),阻礙了其在發(fā)展中國(guó)家的廣泛應(yīng)用。因此,發(fā)展低毒、單劑量及長(zhǎng)期有效的狂犬病疫苗對(duì)控制狂犬病具有重要意義。近來(lái)研究表明,過(guò)表達(dá)樹(shù)突狀細(xì)胞(Dendritic cells,DCs)刺激因子的重組狂犬病病毒可在淋巴結(jié)及外周血中招募并成熟DCs及B淋巴細(xì)胞,進(jìn)而迅速產(chǎn)生顯著高水平的抗病毒中和抗體(Virus-neutralizing antibodies,VNA),但其有效持續(xù)時(shí)間短。當(dāng)前,尚未有研究通過(guò)應(yīng)用免疫記憶的作用機(jī)制來(lái)延長(zhǎng)狂犬病疫苗誘導(dǎo)的體液免疫。本研究通過(guò)反向遺傳技術(shù),在弱毒疫苗株r LBNSE的基礎(chǔ)上過(guò)表達(dá)對(duì)免疫細(xì)胞的生長(zhǎng)、存活及分化方面具有廣泛效應(yīng)的白介素-7(Interleukin-7,IL-7),并拯救獲得重組狂犬病病毒(r LBNSE-IL7)。在確定r LBNSE-IL7的體外生長(zhǎng)特性不因外源基因IL-7的插入而受到影響之后,通過(guò)ELISA檢測(cè)重組病毒感染細(xì)胞上清中IL-7的表達(dá),發(fā)現(xiàn)只有rLBNSE-IL7以劑量依賴形式表達(dá)IL-7,并對(duì)細(xì)胞無(wú)明顯副作用。將r LBNSE-IL7在BSR細(xì)胞中連續(xù)傳代10次,各代次均保持IL-7的穩(wěn)定遺傳。為鑒定IL-7的表達(dá)對(duì)狂犬病病毒致病性的影響,將rLBNSE、rLBNSE-IL7以及陰性對(duì)照重組病毒rLBNSE-IL7(-)腦內(nèi)途徑感染免疫功能完全和不完全的小鼠。我們發(fā)現(xiàn)腦內(nèi)高表達(dá)的IL-7能夠限制病毒在腦內(nèi)的轉(zhuǎn)錄和復(fù)制,進(jìn)一步減弱狂犬病病毒的致病力,而非IL-7的插入造成病毒基因組長(zhǎng)度的增加所導(dǎo)致。為進(jìn)一步分析IL-7對(duì)狂犬病病毒免疫原性的作用,將重組病毒后肢肌肉途徑免疫小鼠,進(jìn)行依賴T細(xì)胞的與B細(xì)胞相互作用的相關(guān)免疫機(jī)制研究。與親本病毒r LBNSE相比,重組病毒表達(dá)的IL-7可以顯著增加淋巴結(jié)中濾泡性T輔助細(xì)胞(T follicular helper cells,Tfh cells)、生發(fā)中心B細(xì)胞(Germinal center cells,GC B cells)、長(zhǎng)壽命記憶性B細(xì)胞(Memory B cells,Bmem)的數(shù)量和骨髓中長(zhǎng)壽命漿細(xì)胞(Plasma cells,PCs)的比例。由單劑量免疫rLBNSE-IL7所增殖的長(zhǎng)壽命PCs可產(chǎn)生更加強(qiáng)大的免疫效力,維持顯著高水平的VNA和相關(guān)抗體亞類(lèi)長(zhǎng)達(dá)360天,且能夠長(zhǎng)期有效保護(hù)絕大多數(shù)小鼠抵抗強(qiáng)毒株攻擊。此外,IL-7所增加的長(zhǎng)壽命Bmem能夠于再次共同免疫r LBNSE后,分化成抗體分泌細(xì)胞,迅速產(chǎn)生更高水平的VNA及相應(yīng)抗體亞類(lèi)。上述結(jié)果表明IL-7促進(jìn)了Tfh細(xì)胞的增殖,并在其協(xié)助下增強(qiáng)了GCs反應(yīng),進(jìn)一步生成與維持免疫記憶相關(guān)的長(zhǎng)壽命Bmem和PCs,從而增強(qiáng)了機(jī)體針對(duì)狂犬病病毒的特異性初次免疫反應(yīng)和再次免疫應(yīng)答。綜上所述,本研究成功構(gòu)建了表達(dá)IL-7的重組狂犬病病毒r LBNSE-IL7,鑒定了外源基因IL-7在病毒基因組中的表達(dá)不影響病毒的體外生長(zhǎng)特性;且腦內(nèi)高表達(dá)的IL-7可以降低狂犬病病毒的致病力;并通過(guò)促進(jìn)GCs中的Tfh細(xì)胞與B細(xì)胞的相關(guān)免疫反應(yīng)從而增殖長(zhǎng)壽命Bmem和PCs以增強(qiáng)狂犬病病毒的免疫記憶,改善初次免疫保護(hù)性反應(yīng)和再次免疫應(yīng)答。此研究結(jié)果表明rLBNSE-IL7具有成為低毒、高效的動(dòng)物狂犬病疫苗的潛能。
[Abstract]:Rabies virus (Rabies) infection as one of the oldest, is composed of rabies virus (Rabies Virus, RABV) zoonotic infectious disease caused by central nervous system, distributed in most parts of the world. China's annual number of deaths in the rabies ranked second in the world and all kinds of infectious diseases before the five, of which more than 95% the cases are caused by direct or indirect contact with diseased or poisonous animal caused. At present, the incidence of rabies after clinical treatment is still a difficult problem in the world, the mortality rate as high as 100%, there is no effective treatment. The data show that more than 70% of the coverage rate of immune dogs can effectively control rabies, therefore. Animal immune prevention is the most effective containment of human rabies. The measures currently used for animal rabies vaccines are inactivated vaccine is safe, but the price is expensive, and needs more time to be immune Have good long-term protection, has hindered its widespread use in the developing world. Therefore, the development of low toxicity, single dose and long-term effective rabies vaccine is important for rabies control. Recent research showed that over expression of dendritic cells (Dendritic, cells, DCs) stimulation of recombinant rabies virus factors in lymph nodes and peripheral blood recruit and mature DCs and B lymphocytes, and then quickly produced significantly higher levels of neutralizing antibody (Virus-neutralizing, antibodies, VNA), but the effective duration is short. At present, there has not been a mechanism through the application of immune memory to extend the humoral immunity induced by rabies vaccine. This study by reverse genetic technique, overexpression of immune cell growth based on attenuated vaccine strain R LBNSE, has a wide effect on survival and differentiation of interleukin -7 (Interleukin-7, IL-7), and. Save the recombinant rabies virus (R LBNSE-IL7) r LBNSE-IL7. After determining the in vitro growth characteristics due to the insertion of exogenous gene IL-7 affected, recombinant virus ELISA infection by detecting the expression of IL-7 in the supernatant, found that only rLBNSE-IL7 expression of IL-7 in a dose dependent form, and no obvious side effects on R LBNSE-IL7 cells. In BSR cells were passaged 10 times and each generation are to maintain the stability of the IL-7 genetic expression. To identify the effect of IL-7 on the pathogenicity of rabies virus, rLBNSE, rLBNSE-IL7 and negative control recombinant virus rLBNSE-IL7 (-) complete and incomplete the immune function of mice infected brain. We found high expression in the brain the IL-7 transcription and replication in the brain to limit the virus, further attenuated rabies virus pathogenicity, rather than the increase of IL-7 due to the insertion of the viral genome length caused by in. Further analysis of the effect of IL-7 on the immunogenicity of rabies virus, the recombinant virus immunized mice by way of hindlimb muscles, dependent T cells and B cells related to immune mechanism of the interaction of LBNSE and R. Compared with the parental virus, the recombinant virus IL-7 can significantly increase the expression of T in lymph node follicular helper cells (T follicular helper cells, Tfh cells), germinal center B cell (Germinal center cells GC, B cells), long-lived memory B cells (Memory B cells, Bmem) and the number of long-lived plasma cells in the bone marrow (Plasma, cells, PCs). The proportion of long-lived PCs proliferation by single dose immunization can be rLBNSE-IL7 have stronger immunity, maintain significantly higher levels of VNA and related antibody subclasses for up to 360 days, and can effectively protect the most mice against virulent attacks. In addition, long-life Bmem added to the IL-7 to another LBNSE R after immunization, differentiate into antibody secreting cells, quickly produce higher levels of VNA and the corresponding subunits. These results indicated that IL-7 promoted the proliferation of Tfh cells and enhanced GCs reaction in its assistance, further generation associated with maintaining immunological memory long life Bmem and PCs, so as to enhance the the specificity of the primary immune response to rabies virus and secondary immune response. In summary, this study successfully constructed IL-7 expressing recombinant rabies virus R LBNSE-IL7, identified the expression of IL-7 gene in the viral genome does not affect the virus in vitro growth characteristics; and in the brain of the high expression of IL-7 can reduce the rabies virus force; and through the immune response in GCs Tfh cells and promote B cells proliferation and long life Bmem and PCs to enhance the rabies virus immune memory, improve primary immune protection The results of this study suggest that rLBNSE-IL7 has the potential to become a low toxic and efficient animal rabies vaccine.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S852.65

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