本文選題：HBV + NK細(xì)胞��； 參考：《中國(guó)科學(xué)技術(shù)大學(xué)》2012年博士論文

【摘要】：HBV屬于嗜肝DNA病毒科,是有包膜病毒并含有大約3.2kb的環(huán)狀雙鏈DNA基因組。HBV感染可導(dǎo)致有或無(wú)臨床表征的肝炎。雖然已經(jīng)研發(fā)出預(yù)防性的高效疫苗,但是全球仍有大約3.5億人是慢性HBV感染者并且有發(fā)展為晚期肝臟疾病以及肝癌的風(fēng)險(xiǎn)。HBV的有效清除依賴于患者的年齡和免疫狀態(tài),免疫應(yīng)答低下導(dǎo)致持續(xù)性或者慢性HBV感染。因此,宿主的免疫應(yīng)答在HBV感染過(guò)程中具有重要的作用。 一直以來(lái),免疫學(xué)家們認(rèn)為適應(yīng)性免疫系統(tǒng)在HBV的清除和致病方面具有重要的作用,相比之下,作為天然免疫系統(tǒng)重要成分的NK細(xì)胞在HBV感染中的作用卻不是很清楚。雖有文獻(xiàn)表明清除小鼠的NK細(xì)胞導(dǎo)致HBV耐受的現(xiàn)象,以及急性HBV患者清除HBV的感染伴隨著體內(nèi)NK細(xì)胞活化的現(xiàn)象,但是對(duì)于HBV感染后NK細(xì)胞如何發(fā)揮抗HBV感染功能則知之甚少。近來(lái),有很多關(guān)于NK細(xì)胞對(duì)適應(yīng)性免疫系統(tǒng)調(diào)節(jié)的報(bào)道。但是HBV感染過(guò)程中NK細(xì)胞是否具有對(duì)適應(yīng)性免疫系統(tǒng)的調(diào)節(jié)功能也不是很清楚。因而,NK細(xì)胞在HBV感染過(guò)程中是否具有重要的作用,以及NK細(xì)胞是否參與對(duì)后續(xù)適應(yīng)性免疫應(yīng)答的調(diào)節(jié),以及兩大免疫系統(tǒng)如何相互協(xié)調(diào)介導(dǎo)HBV的清除?這正是我們所關(guān)心的問(wèn)題。 因此,本論文的研究目的在于研究HBV清除的免疫學(xué)機(jī)制,探討HBV感染過(guò)程中是否需要NK細(xì)胞的參與,以及重點(diǎn)探索NK細(xì)胞是否介導(dǎo)對(duì)適應(yīng)性免疫細(xì)胞的活化,以期為臨床慢性HBV患者的治療以及疫苗的研制提供新的策略。 在本論文中對(duì)小鼠尾靜脈高壓注射pAAV/HBV1.2質(zhì)粒,建立了小鼠急性HBV感染模型。利用該模型我們深入探討了HBV清除的免疫學(xué)機(jī)制以及NK細(xì)胞如何誘導(dǎo)適應(yīng)性免疫系統(tǒng)啟動(dòng)而清HBV感染。根據(jù)CD8-/-小鼠血清HBV相關(guān)抗原的檢測(cè)以及體外阻斷CD8+T細(xì)胞后上清中細(xì)胞因子的分泌情況評(píng)估CD8+T細(xì)胞在HBV清除中的作用；采用流式細(xì)胞術(shù)檢測(cè)NK細(xì)胞的活化,以及利用NK細(xì)胞清除和轉(zhuǎn)輸實(shí)驗(yàn)證明NK細(xì)胞如何參與抗HBV感染；通過(guò)對(duì)缺陷小鼠的特定細(xì)胞清除及過(guò)繼轉(zhuǎn)輸實(shí)驗(yàn)檢測(cè)NK細(xì)胞對(duì)CD8+T細(xì)胞的影響；清除CD4-/-小鼠的NK細(xì)胞研究其是否參與NK細(xì)胞所介導(dǎo)的抗病毒及促進(jìn)CD8+T細(xì)胞的活化；利用轉(zhuǎn)輸實(shí)驗(yàn)及流式細(xì)胞術(shù)檢測(cè)NK細(xì)胞對(duì)CD4+T細(xì)胞的調(diào)節(jié)；通過(guò)抗體阻斷實(shí)驗(yàn)進(jìn)一步證明DCs亦受到NK細(xì)胞的調(diào)節(jié)。另外,通過(guò)肝內(nèi)注射及尾靜脈高壓注射Ad-EGFP實(shí)驗(yàn),以及手術(shù)摘除實(shí)驗(yàn)、體內(nèi)外分析肝臟淋巴結(jié)細(xì)胞等實(shí)驗(yàn)驗(yàn)證肝臟淋巴結(jié)與肝臟的關(guān)系。利用免疫放射法(IRMA)定量檢測(cè)小鼠血清中HBsAg, HBeAg, anti-HBs, HBV DNA以及ALT水平,以及H-E染色和免疫組化分析來(lái)WT小鼠以及缺陷小鼠中病毒的清除特點(diǎn)；CBA檢測(cè)培養(yǎng)上清中細(xì)胞因子的變化；流式細(xì)胞術(shù)檢測(cè)NK細(xì)胞、CD8+T細(xì)胞、CD4+T細(xì)胞以及DCs的表型變化以及胞內(nèi)細(xì)胞因子的表達(dá)情況。通過(guò)上述一系列的研究方法,我們?nèi)〉萌缦碌膶?shí)驗(yàn)結(jié)果： 1.CD8+T細(xì)胞參與無(wú)細(xì)胞病變性在HBV清除過(guò)程中具有重要的作用 通過(guò)對(duì)C57BL/6小鼠高壓尾靜脈注射20μgpAAV/HBV質(zhì)粒,檢測(cè)血清HBsAg, HBeAg, anti-HBs, HBV DNA以及ALT水平。結(jié)果表明HBV在感染小鼠體內(nèi)能夠?qū)崿F(xiàn)HBV的復(fù)制,且表達(dá)HBV相關(guān)抗原,WT小鼠可在HBV感染后3-4周內(nèi)清除病毒感染,但是不引起肝細(xì)胞損傷和淋巴細(xì)胞浸潤(rùn),即為無(wú)細(xì)胞病變性感染模型,類似于人類急性HBV感染。為了進(jìn)一步檢測(cè)HBV的清除是否有免疫系統(tǒng)的參與,我們檢測(cè)CD8-/-小鼠在該模型中的特點(diǎn),提示CD8-/-小鼠血清HBsAg和HBeAg均明顯高于WT小鼠。分離WT小鼠脾臟的淋巴細(xì)胞,體外用a-CD8抗體阻斷CD8+T細(xì)胞的作用,導(dǎo)致脾臟淋巴細(xì)胞IFN-γ及TNF-α分泌能力均明顯降低,上述結(jié)果表明提示CD8+T細(xì)胞對(duì)于急性HBV感染模型中HBV的清除具有重要的作用。 2.NK細(xì)胞來(lái)源的IFN-y促進(jìn)HBV的清除 我們?cè)谠撃Ｐ椭邪l(fā)現(xiàn)HBV質(zhì)粒高壓注射后第3天,脾臟NK細(xì)胞明顯增加,且CD69表達(dá)亦明顯增加。NK細(xì)胞在HBV質(zhì)粒高壓注射后第5天和第7天IFN-γ表達(dá)明顯增加。為進(jìn)一步研究NK細(xì)胞在HBV清除中的作用,我們清除WT小鼠體內(nèi)的NK細(xì)胞,肝組織病理染色結(jié)果表明NK細(xì)胞清除小鼠的肝組織HBcAg表達(dá)以及血清HBsAg及HBeAg表達(dá)明顯高于對(duì)照小鼠。同樣NK細(xì)胞缺陷小鼠Nfi13-/-小鼠在HBV質(zhì)粒高壓注射后亦不能清除HBV感染,而且分別轉(zhuǎn)輸WT小鼠的NK細(xì)胞和GKO小鼠的NK細(xì)胞至Nfi13-/-小鼠,則發(fā)現(xiàn)轉(zhuǎn)輸WT小鼠的NK細(xì)胞其血清HBsAg低于轉(zhuǎn)輸GKO小鼠NK細(xì)胞組。另外對(duì)NK細(xì)胞清除小鼠尾靜脈注射mIFN-γ可以明顯降低NK細(xì)胞清除所導(dǎo)致血清HBsAg的升高。因此,上述結(jié)果表明NK細(xì)胞來(lái)源的IFN-γ是參與HBV清除的主要功能。 3.NK細(xì)胞來(lái)源的IFN-y促進(jìn)抗HBV CD8+T細(xì)胞免疫應(yīng)答 上述結(jié)果提示HBV清除過(guò)程中既需要CD8+T細(xì)胞的參與,又依賴于NK細(xì)胞的作用。那么二者之間是否存在調(diào)節(jié)作用呢?我們清除WT小鼠體內(nèi)NK細(xì)胞,發(fā)現(xiàn)CD8+T細(xì)胞數(shù)目明顯減低；肝臟和脾臟CD8+T細(xì)胞CD44+CD62L-CD8+T表達(dá)以及分泌IFN-γ明顯減低；且NK細(xì)胞清除小鼠的肝臟HBV-特異性CD8+T細(xì)胞的活化明顯減低。為了明確NK細(xì)胞分泌的IFN-γ促進(jìn)CD8+T細(xì)胞的免疫應(yīng)答,我們清除CD8-/-小鼠體內(nèi)NK細(xì)胞,轉(zhuǎn)輸CFSE標(biāo)記的CD8+T細(xì)胞,并同時(shí)向NK細(xì)胞清除CD8-/-小鼠體內(nèi)分別轉(zhuǎn)輸WT小鼠的NK細(xì)胞以及GKO(Ifn-/-)小鼠的NK細(xì)胞,我們發(fā)現(xiàn)CFSE-CD8+T細(xì)胞在清除NK細(xì)胞的受體中的增殖以及分泌IFN-γ的能力與對(duì)照小鼠(不清除NK細(xì)胞組)相比均明顯下降。轉(zhuǎn)輸WT小鼠的NK細(xì)胞則能促進(jìn)CFSE-CD8+T細(xì)胞的增殖以及分泌IFN-γ。相比之下,轉(zhuǎn)輸GKO小鼠NK細(xì)胞其促進(jìn)CFSE-CD8+T細(xì)胞的增殖及分泌IFN-γ的能力相對(duì)較弱。另外,轉(zhuǎn)輸CFSE-CD8+T胞至GKO小鼠體內(nèi),并對(duì)一些受體小鼠轉(zhuǎn)輸WT小鼠的NK細(xì)胞,結(jié)果表明轉(zhuǎn)輸WT小鼠的NK細(xì)胞的受體小鼠,其肝臟和脾臟中CFSE-CD8+T細(xì)胞的增殖及分泌IFN-γ明顯增強(qiáng)。綜上所述,NK細(xì)胞分泌的IFN-γ促進(jìn)CD8+T細(xì)胞的免疫應(yīng)答。 4.NK細(xì)胞促進(jìn)抗HBV-CD8+T細(xì)胞的再次免疫應(yīng)答 接下來(lái),分選HBV質(zhì)粒高壓注射后第5天的脾臟CD8+T細(xì)胞并標(biāo)記CFSE,分別轉(zhuǎn)輸至NK細(xì)胞清除小鼠及對(duì)照小鼠體內(nèi),24小時(shí)后對(duì)受體小鼠高壓注射HBV質(zhì)粒。發(fā)現(xiàn)NK細(xì)胞參于脾臟抗HBV CD8+T細(xì)胞的再次免疫應(yīng)答。為了進(jìn)一步佐證此觀點(diǎn),我們又分選了HBV質(zhì)粒高壓注射后第14天的肝臟CD8+T細(xì)胞并標(biāo)記CFSE,分別轉(zhuǎn)輸至NK細(xì)胞清除小鼠及對(duì)照小鼠體內(nèi)。同樣發(fā)現(xiàn)NK細(xì)胞清除小鼠的肝臟和脾臟中CFSE-CD8+T細(xì)胞的增殖及分泌IFN-γ的能力明顯下降。因此,NK細(xì)胞參于來(lái)自于肝臟或脾臟的抗HBV CD8+T細(xì)胞的再次免疫應(yīng)答。 5.CD4+T細(xì)胞在NK細(xì)胞抗HBV感染及抗HBV CD8+T細(xì)胞活化中的作用 為了檢測(cè)CD4+T細(xì)胞是否在NK細(xì)胞控制HBV的感染及調(diào)節(jié)CD8+T細(xì)胞的活化中發(fā)揮作用,我們檢測(cè)Ragl-/-和CD4-/-小鼠清除HBV的情況,結(jié)果表明Ragl-/-和CD4-/-小鼠體內(nèi)雖然存在NK細(xì)胞,但是其不能發(fā)揮抗HBV作用。進(jìn)一步清除Rag1-/-和CD4-/-小鼠中NK細(xì)胞,發(fā)現(xiàn)血清HBsAg的亦無(wú)明顯變化。而WT小鼠中NK細(xì)胞可控制HBV的感染。進(jìn)一步實(shí)驗(yàn)發(fā)現(xiàn)CD4-/-小鼠體內(nèi)NK細(xì)胞不能促進(jìn)CD8+T細(xì)胞分泌IFN-γ,因?yàn)閃T小鼠的NK細(xì)胞明顯促進(jìn)HBV特異性CD8+T細(xì)胞分泌IFN-γ。清除CD4-/-小鼠體內(nèi)的NK細(xì)胞也不能明顯改變CD4-/-小鼠體內(nèi)CD8+T細(xì)胞分泌IFN-γ。因此我們認(rèn)為CD4-/-小鼠的NK細(xì)胞間接抗HBV作用,以及間接調(diào)節(jié)CD8+T細(xì)胞。且NK細(xì)胞的抗病毒作用依賴于CD4+T細(xì)胞的存在。上述結(jié)果表明,CD4+T細(xì)胞不僅介導(dǎo)NK細(xì)胞的抗HBV感染,也參與其對(duì)CD8+T細(xì)胞的調(diào)節(jié)。 6.CD4+T細(xì)胞輔助產(chǎn)生抗HBV CD8+T細(xì)胞免疫應(yīng)答 上述結(jié)果表明CD4+T細(xì)胞參與HBV的清除,我們進(jìn)一步檢測(cè)CD4-/-小鼠清除HBV的能力,結(jié)果表明CD4-/-小鼠血清HBsAg和HBeAg均明顯高于WT小鼠,而且HBV質(zhì)粒高壓注射后第8周也不能清除HBV感染。提示CD4+T細(xì)胞也參與HBV的清除過(guò)程。轉(zhuǎn)輸小鼠的CD8+T細(xì)胞至Rag1-/-小鼠并不能幫助該小鼠清除HBV感染,提示CD8+T細(xì)胞抗HBV感染需要CD4+T細(xì)胞的輔助。另外,體外阻斷小鼠脾臟CD4+T細(xì)胞的作用,亦導(dǎo)致脾臟淋巴細(xì)胞分泌IFN-y下調(diào)。因此,CD8+T細(xì)胞發(fā)揮抗HBV感染功能需要CD4+T細(xì)胞輔助。 7.NK細(xì)胞促進(jìn)CD4+T細(xì)胞免疫應(yīng)答 既然CD4+T細(xì)胞輔助CD8+T細(xì)胞抗病毒免疫應(yīng)答,而且NK細(xì)胞也促進(jìn)CD8+T細(xì)胞的免疫應(yīng)答。CD4+T細(xì)胞與NK細(xì)胞之間可能存在調(diào)節(jié)關(guān)系。比較CD4-/-小鼠與WT小鼠中NK細(xì)胞的功能,結(jié)果表明CD4-/-小鼠中NK細(xì)胞的功能正常。進(jìn)一步檢測(cè)NK細(xì)胞是否調(diào)節(jié)CD4+T細(xì)胞的功能,轉(zhuǎn)輸CFSE標(biāo)記的CD4+T細(xì)胞至CD4-/-小鼠體內(nèi),發(fā)現(xiàn)NK細(xì)胞清除導(dǎo)致CFSE-CD4+T細(xì)胞在受體小鼠的肝臟和脾臟的增殖及IFN-γ分泌明顯減低。且分離來(lái)自于NK細(xì)胞清除小鼠的肝臟CD4+T細(xì)胞,體外經(jīng)Core129肽段刺激,產(chǎn)生分泌IFN-y的HBV-特異性-CD4+T細(xì)胞的能力亦明顯減低。以上結(jié)果表明NK細(xì)胞不受到CD4+T細(xì)胞的調(diào)節(jié),且NK細(xì)胞促進(jìn)CD4+T細(xì)胞的抗HBV免疫應(yīng)答。 8.NK細(xì)胞分泌的IFN-γ促進(jìn)CDllb+DCs免疫應(yīng)答 NK細(xì)胞清除小鼠脾臟CDllb+DCs明顯減低,且CDllb+DCs表面CD86和CD80的表達(dá)也明顯下降,且脾細(xì)胞IL-18,IL-15和IFN-α的mRNA表達(dá)水平明顯下調(diào)。尾靜脈注射IFN-y以清除小鼠體內(nèi)的IFN-y,發(fā)現(xiàn)該小鼠脾臟CD11b+DCs的表達(dá)亦明顯下降下降,且脾細(xì)胞IL-15和IFN-α的mRNA表達(dá)水平亦下調(diào)。因此,NK細(xì)胞分泌的IFN-y亦促進(jìn)脾臟CD11b+DCs的活化,其可能介導(dǎo)后續(xù)適應(yīng)性免疫應(yīng)答的活化。 9.肝臟淋巴結(jié)對(duì)于HBV免疫應(yīng)答發(fā)生的影響 另外,通過(guò)對(duì)小鼠肝內(nèi)注射以及高壓尾靜脈注射Ad-EGFP,發(fā)現(xiàn)肝臟門(mén)部右側(cè)的淋巴結(jié)可以引流肝臟EFGP+細(xì)胞,提示肝臟淋巴結(jié)引流肝臟的細(xì)胞。并且發(fā)現(xiàn)肝臟淋巴結(jié)能產(chǎn)生抗HBV特異性免疫應(yīng)答。進(jìn)一步實(shí)驗(yàn)表明手術(shù)摘除肝臟淋巴結(jié)導(dǎo)致大約30%的HBV耐受小鼠出現(xiàn),而摘除脾臟卻不影響小鼠HBV感染情況。進(jìn)一步確證了HBV感染模型中肝臟淋巴結(jié)發(fā)生抗HBV的免疫應(yīng)答。檢測(cè)HBV感染后肝臟淋巴結(jié)細(xì)胞表型,發(fā)現(xiàn)HBV感染后CD8+T細(xì)胞及DCs發(fā)生增殖。因此,肝臟淋巴結(jié)可以發(fā)生針對(duì)HBV特異性免疫應(yīng)答。 綜上所述,我們對(duì)成年雄性小鼠高壓尾靜脈注射pAAV/HBV1.2質(zhì)粒,建立模擬人類急性HBV感染的小鼠HBV感染模型。通過(guò)研究我們?cè)敿?xì)闡述了HBV清除的免疫學(xué)機(jī)制,明確了NK細(xì)胞在HBV的清除中的重要作用,證明了NK細(xì)胞分泌的IFN-γ介導(dǎo)了后續(xù)適應(yīng)性免疫的活化進(jìn)而促進(jìn)HBV的清除。另外,我們亦發(fā)現(xiàn)肝臟淋巴結(jié)在HBV感染中具有重要的作用,肝臟淋巴結(jié)能產(chǎn)生抗HBV特異性CD8+T細(xì)胞,為更好的解釋HBV感染中的免疫學(xué)機(jī)制提供理論依據(jù)。
[Abstract]:HBV belongs to the liver of the liver DNA virus, which has a enveloped virus and contains approximately 3.2kb of the cyclic double chain DNA genome.HBV infection that can lead to or without clinical characterization of hepatitis. Although a prophylactic and efficient vaccine has been developed, about 350 million people worldwide are chronic HBV infected and have the development of advanced liver disease and the wind of liver cancer. The effective clearance of risk.HBV depends on the age and immune state of the patient, and the low immune response leads to persistent or chronic HBV infection. Therefore, the host immune response plays an important role in the HBV infection process.
Immunologists have long believed that the adaptive immune system plays an important role in the scavenging and pathogenesis of HBV. In contrast, the role of NK cells as an important component of the natural immune system is not very clear in HBV infection. Although the literature shows that the elimination of NK cells in mice leads to HBV tolerance and the acute HBV patients. The infection of HBV is associated with the activation of NK cells in the body, but little is known about how NK cells play an anti HBV infection after HBV infection. Recently, there are many reports about the regulation of the adaptive immune system of NK cells. But whether NK cells have the function of regulating the adaptive immune system in the course of HBV infection is not the same as that of the adaptive immune system. It is clear, therefore, whether NK cells have an important role in HBV infection, and whether NK cells are involved in the regulation of subsequent adaptive immune responses, and how the two major immune systems coordinate with each other to mediate the clearance of HBV, which is what we are concerned about.
Therefore, the purpose of this study is to study the immunological mechanism of HBV scavenging, to explore the need for the participation of NK cells in the process of HBV infection, and to explore whether NK cells can mediate the activation of adaptive immune cells, in order to provide a new strategy for the treatment of chronic HBV patients and the development of the vaccine.
In this paper, pAAV/HBV1.2 plasmids were injected into the tail vein of mice, and a mouse model of acute HBV infection was established. Using this model, we explored the immunological mechanism of HBV clearance and how the NK cells induce the initiation of the adaptive immune system to clear the HBV infection. The detection of HBV related antigens in the serum of CD8-/- mice and the blocking of C in vitro The secretion of cytokines in the supernatant of D8+T cells was used to evaluate the role of CD8+T cells in the clearance of HBV. Flow cytometry was used to detect the activation of NK cells and the use of NK cell clearance and transfer experiments to demonstrate how NK cells were involved in anti HBV infection; NK cells were detected by specific cell scavenging and adoptive transfer experiments on the defective mice. The effect on CD8+T cells; scavenging the NK cells of CD4-/- mice to investigate whether it participates in the anti-virus mediated by NK cells and promotes the activation of CD8+T cells; the transfer test and flow cytometry are used to detect the regulation of NK cells to CD4+T cells; and the antibody blocking experiment further proves that DCs is also regulated by NK cells. In addition, through the liver, the liver is regulated by the liver. The relationship between liver lymph nodes and liver was verified by Ad-EGFP experiment with internal injection and high pressure of caudal vein, operation extirpation and liver lymph node cells. The level of HBsAg, HBeAg, anti-HBs, HBV DNA and ALT in the serum of mice was detected by immuno radiation (IRMA), and H-E staining and immunohistochemical analysis were used for WT. The scavenging characteristics of virus in mice and deficient mice; the change of cytokine in the culture supernatant by CBA; flow cytometry to detect NK cells, CD8+T cells, CD4+T cells and the phenotype changes of DCs and the expression of intracellular cytokines.
1.CD8+T cells play an important role in the scavenging process of HBV.
The serum HBsAg, HBeAg, anti-HBs, HBV DNA and ALT levels were detected by injecting 20 mu gpAAV/HBV plasmids into the high pressure tail vein of C57BL/6 mice. The results showed that HBV could realize the replication of HBV and expressed HBV antigen in the infected mice. The WT mice could remove the virus infection within 3-4 weeks after the infection, but did not cause liver cell damage and drench. In order to further detect the involvement of the immune system in the clearance of HBV, we detected the characteristics of the CD8-/- mice in this model, suggesting that the serum HBsAg and HBeAg of the CD8-/- mice were significantly higher than those of the WT mice in order to further detect the involvement of the immune system in the scavenging of the CD8-/- mice. The lymphocyte of the spleen of WT mice was separated from the lymphocytes of the WT mice. The effect of a-CD8 antibody on CD8+T cells was blocked by external use, and the secretion of IFN- gamma and TNF- alpha in the spleen lymphocytes were significantly reduced. The results suggest that CD8+T cells play an important role in the clearance of HBV in the acute HBV infection model.
2.NK cell derived IFN-y promotes the clearance of HBV
In this model, we found that the spleen NK cells increased obviously at third days after high pressure injection of HBV plasmids, and the expression of CD69 increased obviously in.NK cells in fifth days and seventh days after HBV plasmids injection. To further study the role of NK cells in HBV clearance, we scavenging NK cells in the WT mice and pathological staining of liver tissue. The color results showed that the expression of HBcAg in the liver tissue of NK cells and the expression of HBsAg and HBeAg in the serum were significantly higher than those of the control mice. Similarly, the Nfi13-/- mice of the NK cell defect mice could not clear the HBV infection after the HBV plasmid high pressure injection, and then transferred to the NK cells of the WT mice and the NK cells of the GKO mice to the mice. The serum HBsAg of NK cells in WT mice was lower than that of the GKO mouse NK cell group. In addition, the increase of serum HBsAg caused by the clearance of NK cells by mIFN- gamma in the tail vein of NK cells was significantly reduced. Therefore, the above results showed that IFN- gamma of NK cell sources was the main function of participation in HBV clearance.
3.NK cell derived IFN-y promotes immune response to HBV CD8+T cells
These results suggest that HBV scavenging process requires the involvement of CD8+T cells and the role of NK cells. Then, is there any regulatory effect between the two? We scavenging NK cells in WT mice, finding the number of CD8+T cells significantly reduced, and the CD62L-CD8+T expression of CD44+ in the liver and spleen CD8+T cells and the decrease of IFN- gamma; and In order to clear the IFN- gamma secreted by NK cells to promote the immune response of CD8+T cells, we scavenged NK cells in the CD8-/- mice and transferred CFSE labeled CD8+T cells to clear the IFN- gamma secreted by NK cells. Meanwhile, we removed the CFSE labeled CD8+T cells from the CD8-/- mice and removed the CD8+T cells from the CD8-/- mice to the NK cells. Fn-/-) NK cells in mice, we found that the proliferation of CFSE-CD8+T cells in the scavenging of NK cells and the ability to secrete IFN- gamma were significantly lower than those of the control mice (not scavenging NK cells). The NK cells transferred from WT mice could promote the proliferation of CFSE-CD8+T cells and secrete IFN- gamma. The proliferation of CFSE-CD8+T cells and the ability to secrete IFN- gamma were relatively weak. In addition, the transfer of CFSE-CD8+T cells to GKO mice and the transfer of some receptor mice to NK cells of WT mice showed that the proliferation and secretion of IFN- gamma in the liver and spleen of the recipient mice of the NK cells in the liver and spleen were significantly enhanced. The IFN- gamma secreted by NK cells promotes the immune response of CD8+T cells.
4.NK cells promote reimmunization against HBV-CD8+T cells
Then, the splenic CD8+T cells were selected for fifth days after high pressure injection of HBV plasmids and labeled with CFSE, then transferred to NK cells to clear the mice and the control mice. After 24 hours, the HBV plasmid was injected into the recipient mice by high pressure. It was found that NK cells were used to re immunization of the spleen against HBV CD8+T cells. In order to further support this view, we were also selected. The liver CD8+T cells of fourteenth days after high pressure injection of HBV plasmid were labeled with CFSE and transferred to NK cells to scavenging mice and control mice respectively. It was also found that NK cells cleared the proliferation of CFSE-CD8+T cells and the ability to secrete IFN- gamma in the liver and spleen of mice. Therefore, NK cells were involved in the anti HBV CD8 from the liver and spleen. The immune response of +T cells.
Role of 5.CD4+T cells in NK cells against HBV infection and HBV CD8+T cell activation
In order to detect whether CD4+T cells play a role in controlling HBV infection in NK cells and regulating the activation of CD8+T cells, we detected the scavenging of HBV in Ragl-/- and CD4-/- mice. The results showed that although NK cells existed in Ragl-/- and CD4-/- mice, they could not play an anti HBV act. It was found that there was no significant change in serum HBsAg, while NK cells in WT mice could control the infection of HBV. Further experiments found that NK cells in CD4-/- mice did not promote the secretion of IFN- gamma in CD8+T cells, because NK cells in WT mice obviously promoted the secretion of HBV CD8+T cells. - / - / - CD8+T cells secreted IFN- gamma. Therefore, we think that the NK cells in CD4-/- mice are indirect anti HBV and indirectly regulate CD8+T cells. And the antiviral effect of NK cells depends on the existence of CD4+T cells. The results show that CD4+T cells not only mediate the HBV dyeing of NK cells, but also participate in the regulation of CD8+T cells.
6.CD4+T cells assist in producing anti HBV CD8+T cell immune responses
The above results showed that CD4+T cells were involved in the clearance of HBV. We further detected the ability of CD4-/- mice to scavenge HBV. The results showed that the serum HBsAg and HBeAg in the CD4-/- mice were significantly higher than those of WT mice, and the HBV plasmid could not clear the HBV infection for eighth weeks after the high pressure injection of the HBV plasmid. Cell to Rag1-/- mice did not help the mice to scavenging HBV infection, suggesting that the anti HBV infection of CD8+T cells needed CD4+T cell assistance. In addition, blocking the role of CD4+T cells in the spleen of mice in vitro also resulted in the secretion of IFN-y in the spleen lymphocytes. Therefore, CD8+T cells needed CD4+T cell assistance to resist HBV infection.
7.NK cells promote CD4+T cell immune response
Since CD4+T cells assist the immune response of CD8+T cells, and NK cells also promote the immune response of CD8+T cells, there may be a regulatory relationship between.CD4+T and NK cells. Compare the function of NK cells in CD4-/- mice and WT mice. The results show that the work of NK cells in CD4-/- mice is normal. The function of T cells, transfer of CFSE labeled CD4+T cells to CD4-/- mice, it was found that NK cell clearance resulted in the proliferation of CFSE-CD4+T cells in the liver and spleen and the secretion of IFN- gamma in the receptor mice. The separation from NK cells removed the liver CD4+T cells from mice and stimulated by Core129 peptide segment in vitro, producing IFN-y HBV- specificity. The ability of sex -CD4+T cells was also significantly reduced. The above results showed that NK cells were not regulated by CD4+T cells, and NK cells promoted the anti HBV immune response of CD4+T cells.
IFN- cells secreted by 8.NK cells promote CDllb+DCs immune response
The expression of CDllb+DCs in the spleen of mice decreased significantly by NK cells, and the expression of CD86 and CD80 on the CDllb+DCs surface also decreased obviously, and the mRNA expression level of IL-18, IL-15 and IFN- a in the spleen cells decreased obviously. The tail vein was injected with IFN-y to clear the IFN-y in the mice. The expression level of N- mRNA is also down regulated. Therefore, the IFN-y secreted by NK cells also promotes the activation of CD11b+DCs in the spleen, which may mediate the activation of adaptive immune response.
9. effects of hepatic lymph nodes on HBV immune response
In addition, the lymph nodes on the right side of the hepatic portal were found to drain the liver EFGP+ cells on the right side of the hepatic portal, suggesting that the liver lymph nodes drain the liver cells by using the intrahepatic injection of the liver and the high pressure tail vein for Ad-EGFP. The liver lymph nodes can produce the anti HBV specific immune response. About 30% of the HBV tolerant mice appeared, but the spleen did not affect the HBV infection in mice.

【學(xué)位授予單位】：中國(guó)科學(xué)技術(shù)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 陳月;陳洪濤;吳詩(shī)品;周雅瑩;方愛(ài)萍;;阿德福韋酯對(duì)慢性乙型肝炎患者外周血樹(shù)突狀細(xì)胞和淋巴細(xì)胞亞群的影響[J];廣東醫(yī)學(xué);2010年01期

2 李莉,王元和,張玲珍,胡棟平,張浩,連兆瑞,孔憲濤;消化道腫瘤浸潤(rùn)淋巴細(xì)胞的提取、培養(yǎng)及表型分析[J];現(xiàn)代免疫學(xué);1993年02期

3 黃蓓，，車東媛，張婉蓉;缺氧內(nèi)皮細(xì)胞培養(yǎng)液對(duì)肺動(dòng)脈平滑肌細(xì)胞表型的影響[J];同濟(jì)醫(yī)科大學(xué)學(xué)報(bào);1996年01期

4 魏虎來(lái),裴新燕,姚小健;T-AK細(xì)胞的形態(tài)學(xué)和免疫表型研究[J];中國(guó)免疫學(xué)雜志;1998年04期

5 吳銘,孫雄飛,徐肇明,張新友,李富榮,王興根,陳曉琳,林海清,文鴻光,孫璇,宋通微;基于細(xì)胞表型異常的流式細(xì)胞術(shù)檢測(cè)急性前體B細(xì)胞白血病微小殘留病[J];中國(guó)實(shí)驗(yàn)血液學(xué)雜志;2005年04期

6 段玉芹;程立軍;郭蘭濤;張素欣;陳彥平;陳中;;口腔癌患者外周血樹(shù)突狀細(xì)胞的體外培養(yǎng)及表型分析[J];實(shí)用口腔醫(yī)學(xué)雜志;2007年06期

7 魏開(kāi)鵬;潘興南;王崇國(guó);;兩種成人骨髓間充質(zhì)干細(xì)胞體外分離方法的比較研究[J];實(shí)用肝臟病雜志;2011年02期

8 馮明利,雍宜民,沈惠良,胡懷健;bFGF對(duì)體外培養(yǎng)的兔軟骨細(xì)胞的膠原合成和穩(wěn)定細(xì)胞表型的作用[J];首都醫(yī)科大學(xué)學(xué)報(bào);2001年01期

9 秦建平;蔣明德;;肝星狀細(xì)胞的表型及調(diào)控與肝纖維化[J];世界華人消化雜志;2001年07期

10 程欣;體外腫瘤細(xì)胞引起的樹(shù)突狀細(xì)胞表型和移動(dòng)性的變化[J];國(guó)外醫(yī)學(xué).免疫學(xué)分冊(cè);2002年06期

相關(guān)會(huì)議論文 前10條

1 ;傳染免疫[A];第七屆全國(guó)免疫學(xué)學(xué)術(shù)大會(huì)論文集[C];2010年

2 吳微微;王球達(dá);葛錫銳;;抗CD_3、PHA-P及IL-2對(duì)LAK細(xì)胞培養(yǎng)的生長(zhǎng)特性,細(xì)胞表型的影響[A];中國(guó)細(xì)胞生物學(xué)學(xué)會(huì)第五次會(huì)議論文摘要匯編[C];1992年

3 王紅潔;;K562、U937細(xì)胞中過(guò)表達(dá)PTPalpha引起的細(xì)胞表型變化和基因、蛋白表達(dá)的差異[A];華東六省一市生物化學(xué)與分子生物學(xué)學(xué)會(huì)2006年學(xué)術(shù)交流會(huì)論文集[C];2006年

4 ;自身免疫病[A];第七屆全國(guó)免疫學(xué)學(xué)術(shù)大會(huì)論文集[C];2010年

5 陳成;朱一蓓;;B7-H3分子上調(diào)性表達(dá)在CD40活化的樹(shù)突狀細(xì)胞介導(dǎo)抗腫瘤免疫應(yīng)答中的作用[A];蘇州市自然科學(xué)優(yōu)秀學(xué)術(shù)論文匯編(2008-2009)[C];2010年

6 楊曉紅;李華;柳惠圖;;蛋白激酶C βⅠ亞類的過(guò)表達(dá)對(duì)NRK細(xì)胞表型及生長(zhǎng)的影響[A];中國(guó)細(xì)胞生物學(xué)學(xué)會(huì)第五次會(huì)議論文摘要匯編[C];1992年

7 祁振強(qiáng);胡廣大;楊照華;張福恩;;一種基于免疫反饋原理的新型控制算法[A];2003中國(guó)控制與決策學(xué)術(shù)年會(huì)論文集[C];2003年

8 袁仕善;吳少庭;張仁利;高世同;黃達(dá)娜;余新炳;;重組弓形蟲(chóng)GRA8的表達(dá)及其誘導(dǎo)的免疫應(yīng)答[A];全國(guó)新出現(xiàn)傳染病學(xué)術(shù)研討會(huì)資料匯編[C];2004年

9 江宏兵;田衛(wèi)東;陳希哲;湯煒;劉磊;李曉東;;誘導(dǎo)顱神經(jīng)嵴向第一鰓弓外胚間充質(zhì)細(xì)胞分化的實(shí)驗(yàn)研究[A];2004年中國(guó)口腔頜面修復(fù)重建外科學(xué)術(shù)會(huì)議論文匯編[C];2004年

10 蔣敬庭;吳昌平;鄧海峰;陸明洋;李敏;徐斌;王赫;吳駿;季枚;趙偉慶;Ning Xu;Peter Nilsson-Ehle;;CIK細(xì)胞技術(shù)平臺(tái)的建立及臨床應(yīng)用[A];第九屆全國(guó)腫瘤生物治療學(xué)術(shù)會(huì)議論文集[C];2006年

相關(guān)重要報(bào)紙文章 前10條

1 劉燕玲;免疫辨證指導(dǎo)乙肝治療[N];健康報(bào);2005年

2 陳操 趙玉軍;操縱“主犯”：變異毒株及其“特異”功能[N];中國(guó)畜牧獸醫(yī)報(bào);2007年

3 楊金志;我國(guó)科學(xué)家發(fā)現(xiàn)抑制敗血癥休克的新機(jī)制[N];大眾科技報(bào);2008年

4 余志平;抗RSV感染研究聚焦免疫應(yīng)答[N];中國(guó)醫(yī)藥報(bào);2005年

5 錢(qián)錚;抑制性T細(xì)胞抑制免疫應(yīng)答的機(jī)制被發(fā)現(xiàn)[N];醫(yī)藥經(jīng)濟(jì)報(bào);2007年

6 記者　 呂賢如 范又;我國(guó)艾滋病疫苗I期臨床研究取得重大階段性進(jìn)展[N];光明日?qǐng)?bào);2006年

7 記者 項(xiàng)錚;流感大流行疫苗研制獲重大突破[N];科技日?qǐng)?bào);2007年

8 朱立平;治療關(guān)鍵：修復(fù)和重建抗乙肝病毒免疫[N];健康報(bào);2005年

9 浙江省奉化市蕭王廟街道獸醫(yī)站 王永存;淺談疫苗免疫失敗的原因及對(duì)策②[N];河北農(nóng)民報(bào);2006年

10 鄭靈巧;艾滋病研究“八面”出擊[N];健康報(bào);2007年

相關(guān)博士學(xué)位論文 前10條

1 盧燕來(lái);人γδT細(xì)胞對(duì)A型流感病毒血凝素蛋白及假病毒的免疫應(yīng)答研究[D];北京協(xié)和醫(yī)學(xué)院;2011年

2 鮑嫣;新型B細(xì)胞亞群的發(fā)現(xiàn)及其免疫調(diào)控功能與機(jī)制的研究[D];第二軍醫(yī)大學(xué);2010年

3 張寧;邊緣群細(xì)胞在肝癌發(fā)生和轉(zhuǎn)移中的作用研究[D];第四軍醫(yī)大學(xué);2010年

4 萬(wàn)亞鋒;肝細(xì)胞癌AFPmRNA轉(zhuǎn)染CD40配體活化的B細(xì)胞誘導(dǎo)抗腫瘤免疫的實(shí)驗(yàn)研究[D];華中科技大學(xué);2010年

5 楊麗娟;Th17細(xì)胞抗腫瘤作用及其機(jī)制研究[D];河北醫(yī)科大學(xué);2010年

6 周瑞;H-T-SCID嵌合體動(dòng)物模型中EBV誘導(dǎo)的CD8~+NKT細(xì)胞抗EBV相關(guān)腫瘤研究[D];武漢大學(xué);2010年

7 王克玲;FOXP3基因過(guò)表達(dá)腺病毒轉(zhuǎn)染未成熟樹(shù)突狀細(xì)胞對(duì)實(shí)驗(yàn)性變態(tài)反應(yīng)性腦脊髓炎免疫耐受作用的研究[D];河北醫(yī)科大學(xué);2011年

8 孫健;中國(guó)地區(qū)2371例淋巴組織腫瘤分布特點(diǎn)的分析及原發(fā)性腸道T細(xì)胞及NK細(xì)胞淋巴瘤的臨床病理及分子生物學(xué)特點(diǎn)的研究[D];北京協(xié)和醫(yī)學(xué)院;2011年

9 劉濤;γδT細(xì)胞在幽門(mén)螺桿菌感染中的作用特點(diǎn)及機(jī)制研究[D];第三軍醫(yī)大學(xué);2012年

10 郝健磊;Vγ1γδT細(xì)胞在腫瘤免疫中的調(diào)節(jié)作用及機(jī)制研究[D];南開(kāi)大學(xué);2012年

相關(guān)碩士學(xué)位論文 前10條

1 劉曉芳;細(xì)胞間接觸在誘導(dǎo)骨髓間充質(zhì)干細(xì)胞向平滑肌細(xì)胞分化中作用的實(shí)驗(yàn)研究[D];第三軍醫(yī)大學(xué);2010年

2 季亞娟;冠心病患者外周血T細(xì)胞亞群和NK細(xì)胞的變化相關(guān)性及臨床意義研究[D];安徽醫(yī)科大學(xué);2010年

3 任玲玲;TLR3和TLR7/8對(duì)小鼠妊娠子宮免疫功能的影響機(jī)制研究[D];暨南大學(xué);2011年

4 張艷;CHL瘤細(xì)胞與背景CD4~+T細(xì)胞關(guān)系的研究[D];南方醫(yī)科大學(xué);2010年

5 劉華;胸腺基質(zhì)淋巴生成素對(duì)肺癌患者輔助性T細(xì)胞極化的影響[D];天津醫(yī)科大學(xué);2010年

6 胡林昆;腎移植受者外周血調(diào)節(jié)性T細(xì)胞及其亞群的臨床研究[D];復(fù)旦大學(xué);2010年

7 黃方;結(jié)核菌素純蛋白衍生物刺激人γδT細(xì)胞效應(yīng)分析[D];重慶醫(yī)科大學(xué);2011年

8 崔瑩瑩;凍融抗原負(fù)載的DC-CIK細(xì)胞對(duì)SKOV3的殺傷作用[D];山東大學(xué);2010年

9 陶盛能;CD4~+CD25~+調(diào)節(jié)性T細(xì)胞和再生障礙性貧血之間的關(guān)系[D];安徽醫(yī)科大學(xué);2010年

10 丁景弦;Huh7細(xì)胞中邊緣群細(xì)胞分選及其增殖分裂能力的研究[D];南京醫(yī)科大學(xué);2010年

本文編號(hào)：1789514