本文選題：HBV + NK細胞　； 參考：《中國科學技術(shù)大學》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感染可導致有或無臨床表征的肝炎。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險。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感染過程中具有重要的作用。 一直以來,免疫學家們認為適應(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細胞在HBV感染中的作用卻不是很清楚。雖有文獻表明清除小鼠的NK細胞導致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細胞活化的現(xiàn)象,但是對于HBV感染后NK細胞如何發(fā)揮抗HBV感染功能則知之甚少。近來,有很多關(guān)于NK細胞對適應(yīng)性免疫系統(tǒng)調(diào)節(jié)的報道。但是HBV感染過程中NK細胞是否具有對適應(yīng)性免疫系統(tǒng)的調(diào)節(jié)功能也不是很清楚。因而,NK細胞在HBV感染過程中是否具有重要的作用,以及NK細胞是否參與對后續(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HBV的清除?這正是我們所關(guān)心的問題。 因此,本論文的研究目的在于研究HBV清除的免疫學機制,探討HBV感染過程中是否需要NK細胞的參與,以及重點探索NK細胞是否介導對適應(yīng)性免疫細胞的活化,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清除的免疫學機制以及NK細胞如何誘導適應(yīng)性免疫系統(tǒng)啟動而清HBV感染。根據(jù)CD8-/-小鼠血清HBV相關(guān)抗原的檢測以及體外阻斷CD8+T細胞后上清中細胞因子的分泌情況評估CD8+T細胞在HBV清除中的作用；采用流式細胞術(shù)檢測NK細胞的活化,以及利用NK細胞清除和轉(zhuǎn)輸實驗證明NK細胞如何參與抗HBV感染；通過對缺陷小鼠的特定細胞清除及過繼轉(zhuǎn)輸實驗檢測NK細胞對CD8+T細胞的影響；清除CD4-/-小鼠的NK細胞研究其是否參與NK細胞所介導的抗病毒及促進CD8+T細胞的活化；利用轉(zhuǎn)輸實驗及流式細胞術(shù)檢測NK細胞對CD4+T細胞的調(diào)節(jié)；通過抗體阻斷實驗進一步證明DCs亦受到NK細胞的調(diào)節(jié)。另外,通過肝內(nèi)注射及尾靜脈高壓注射Ad-EGFP實驗,以及手術(shù)摘除實驗、體內(nèi)外分析肝臟淋巴結(jié)細胞等實驗驗證肝臟淋巴結(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WT小鼠以及缺陷小鼠中病毒的清除特點；CBA檢測培養(yǎng)上清中細胞因子的變化；流式細胞術(shù)檢測NK細胞、CD8+T細胞、CD4+T細胞以及DCs的表型變化以及胞內(nèi)細胞因子的表達情況。通過上述一系列的研究方法,我們?nèi)〉萌缦碌膶嶒灲Y(jié)果： 1.CD8+T細胞參與無細胞病變性在HBV清除過程中具有重要的作用 通過對C57BL/6小鼠高壓尾靜脈注射20μgpAAV/HBV質(zhì)粒,檢測血清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,但是不引起肝細胞損傷和淋巴細胞浸潤,即為無細胞病變性感染模型,類似于人類急性HBV感染。為了進一步檢測HBV的清除是否有免疫系統(tǒng)的參與,我們檢測CD8-/-小鼠在該模型中的特點,提示CD8-/-小鼠血清HBsAg和HBeAg均明顯高于WT小鼠。分離WT小鼠脾臟的淋巴細胞,體外用a-CD8抗體阻斷CD8+T細胞的作用,導致脾臟淋巴細胞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細胞對于急性HBV感染模型中HBV的清除具有重要的作用。 2.NK細胞來源的IFN-y促進HBV的清除 我們在該模型中發(fā)現(xiàn)HBV質(zhì)粒高壓注射后第3天,脾臟NK細胞明顯增加,且CD69表達亦明顯增加。NK細胞在HBV質(zhì)粒高壓注射后第5天和第7天IFN-γ表達明顯增加。為進一步研究NK細胞在HBV清除中的作用,我們清除WT小鼠體內(nèi)的NK細胞,肝組織病理染色結(jié)果表明NK細胞清除小鼠的肝組織HBcAg表達以及血清HBsAg及HBeAg表達明顯高于對照小鼠。同樣NK細胞缺陷小鼠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細胞和GKO小鼠的NK細胞至Nfi13-/-小鼠,則發(fā)現(xiàn)轉(zhuǎn)輸WT小鼠的NK細胞其血清HBsAg低于轉(zhuǎn)輸GKO小鼠NK細胞組。另外對NK細胞清除小鼠尾靜脈注射mIFN-γ可以明顯降低NK細胞清除所導致血清HBsAg的升高。因此,上述結(jié)果表明NK細胞來源的IFN-γ是參與HBV清除的主要功能。 3.NK細胞來源的IFN-y促進抗HBV CD8+T細胞免疫應(yīng)答 上述結(jié)果提示HBV清除過程中既需要CD8+T細胞的參與,又依賴于NK細胞的作用。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細胞,發(fā)現(xiàn)CD8+T細胞數(shù)目明顯減低；肝臟和脾臟CD8+T細胞CD44+CD62L-CD8+T表達以及分泌IFN-γ明顯減低；且NK細胞清除小鼠的肝臟HBV-特異性CD8+T細胞的活化明顯減低。為了明確NK細胞分泌的IFN-γ促進CD8+T細胞的免疫應(yīng)答,我們清除CD8-/-小鼠體內(nèi)NK細胞,轉(zhuǎn)輸CFSE標記的CD8+T細胞,并同時向NK細胞清除CD8-/-小鼠體內(nèi)分別轉(zhuǎn)輸WT小鼠的NK細胞以及GKO(Ifn-/-)小鼠的NK細胞,我們發(fā)現(xiàn)CFSE-CD8+T細胞在清除NK細胞的受體中的增殖以及分泌IFN-γ的能力與對照小鼠(不清除NK細胞組)相比均明顯下降。轉(zhuǎn)輸WT小鼠的NK細胞則能促進CFSE-CD8+T細胞的增殖以及分泌IFN-γ。相比之下,轉(zhuǎn)輸GKO小鼠NK細胞其促進CFSE-CD8+T細胞的增殖及分泌IFN-γ的能力相對較弱。另外,轉(zhuǎn)輸CFSE-CD8+T胞至GKO小鼠體內(nèi),并對一些受體小鼠轉(zhuǎn)輸WT小鼠的NK細胞,結(jié)果表明轉(zhuǎn)輸WT小鼠的NK細胞的受體小鼠,其肝臟和脾臟中CFSE-CD8+T細胞的增殖及分泌IFN-γ明顯增強。綜上所述,NK細胞分泌的IFN-γ促進CD8+T細胞的免疫應(yīng)答。 4.NK細胞促進抗HBV-CD8+T細胞的再次免疫應(yīng)答 接下來,分選HBV質(zhì)粒高壓注射后第5天的脾臟CD8+T細胞并標記CFSE,分別轉(zhuǎn)輸至NK細胞清除小鼠及對照小鼠體內(nèi),24小時后對受體小鼠高壓注射HBV質(zhì)粒。發(fā)現(xiàn)NK細胞參于脾臟抗HBV CD8+T細胞的再次免疫應(yīng)答。為了進一步佐證此觀點,我們又分選了HBV質(zhì)粒高壓注射后第14天的肝臟CD8+T細胞并標記CFSE,分別轉(zhuǎn)輸至NK細胞清除小鼠及對照小鼠體內(nèi)。同樣發(fā)現(xiàn)NK細胞清除小鼠的肝臟和脾臟中CFSE-CD8+T細胞的增殖及分泌IFN-γ的能力明顯下降。因此,NK細胞參于來自于肝臟或脾臟的抗HBV CD8+T細胞的再次免疫應(yīng)答。 5.CD4+T細胞在NK細胞抗HBV感染及抗HBV CD8+T細胞活化中的作用 為了檢測CD4+T細胞是否在NK細胞控制HBV的感染及調(diào)節(jié)CD8+T細胞的活化中發(fā)揮作用,我們檢測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細胞,但是其不能發(fā)揮抗HBV作用。進一步清除Rag1-/-和CD4-/-小鼠中NK細胞,發(fā)現(xiàn)血清HBsAg的亦無明顯變化。而WT小鼠中NK細胞可控制HBV的感染。進一步實驗發(fā)現(xiàn)CD4-/-小鼠體內(nèi)NK細胞不能促進CD8+T細胞分泌IFN-γ,因為WT小鼠的NK細胞明顯促進HBV特異性CD8+T細胞分泌IFN-γ。清除CD4-/-小鼠體內(nèi)的NK細胞也不能明顯改變CD4-/-小鼠體內(nèi)CD8+T細胞分泌IFN-γ。因此我們認為CD4-/-小鼠的NK細胞間接抗HBV作用,以及間接調(diào)節(jié)CD8+T細胞。且NK細胞的抗病毒作用依賴于CD4+T細胞的存在。上述結(jié)果表明,CD4+T細胞不僅介導NK細胞的抗HBV感染,也參與其對CD8+T細胞的調(diào)節(jié)。 6.CD4+T細胞輔助產(chǎn)生抗HBV CD8+T細胞免疫應(yīng)答 上述結(jié)果表明CD4+T細胞參與HBV的清除,我們進一步檢測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細胞也參與HBV的清除過程。轉(zhuǎn)輸小鼠的CD8+T細胞至Rag1-/-小鼠并不能幫助該小鼠清除HBV感染,提示CD8+T細胞抗HBV感染需要CD4+T細胞的輔助。另外,體外阻斷小鼠脾臟CD4+T細胞的作用,亦導致脾臟淋巴細胞分泌IFN-y下調(diào)。因此,CD8+T細胞發(fā)揮抗HBV感染功能需要CD4+T細胞輔助。 7.NK細胞促進CD4+T細胞免疫應(yīng)答 既然CD4+T細胞輔助CD8+T細胞抗病毒免疫應(yīng)答,而且NK細胞也促進CD8+T細胞的免疫應(yīng)答。CD4+T細胞與NK細胞之間可能存在調(diào)節(jié)關(guān)系。比較CD4-/-小鼠與WT小鼠中NK細胞的功能,結(jié)果表明CD4-/-小鼠中NK細胞的功能正常。進一步檢測NK細胞是否調(diào)節(jié)CD4+T細胞的功能,轉(zhuǎn)輸CFSE標記的CD4+T細胞至CD4-/-小鼠體內(nèi),發(fā)現(xiàn)NK細胞清除導致CFSE-CD4+T細胞在受體小鼠的肝臟和脾臟的增殖及IFN-γ分泌明顯減低。且分離來自于NK細胞清除小鼠的肝臟CD4+T細胞,體外經(jīng)Core129肽段刺激,產(chǎn)生分泌IFN-y的HBV-特異性-CD4+T細胞的能力亦明顯減低。以上結(jié)果表明NK細胞不受到CD4+T細胞的調(diào)節(jié),且NK細胞促進CD4+T細胞的抗HBV免疫應(yīng)答。 8.NK細胞分泌的IFN-γ促進CDllb+DCs免疫應(yīng)答 NK細胞清除小鼠脾臟CDllb+DCs明顯減低,且CDllb+DCs表面CD86和CD80的表達也明顯下降,且脾細胞IL-18,IL-15和IFN-α的mRNA表達水平明顯下調(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的表達亦明顯下降下降,且脾細胞IL-15和IFN-α的mRNA表達水平亦下調(diào)。因此,NK細胞分泌的IFN-y亦促進脾臟CD11b+DCs的活化,其可能介導后續(xù)適應(yīng)性免疫應(yīng)答的活化。 9.肝臟淋巴結(jié)對于HBV免疫應(yīng)答發(fā)生的影響 另外,通過對小鼠肝內(nèi)注射以及高壓尾靜脈注射Ad-EGFP,發(fā)現(xiàn)肝臟門部右側(cè)的淋巴結(jié)可以引流肝臟EFGP+細胞,提示肝臟淋巴結(jié)引流肝臟的細胞。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。進一步實驗表明手術(shù)摘除肝臟淋巴結(jié)導致大約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HBV感染后肝臟淋巴結(jié)細胞表型,發(fā)現(xiàn)HBV感染后CD8+T細胞及DCs發(fā)生增殖。因此,肝臟淋巴結(jié)可以發(fā)生針對HBV特異性免疫應(yīng)答。 綜上所述,我們對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。通過研究我們詳細闡述了HBV清除的免疫學機制,明確了NK細胞在HBV的清除中的重要作用,證明了NK細胞分泌的IFN-γ介導了后續(xù)適應(yīng)性免疫的活化進而促進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細胞,為更好的解釋HBV感染中的免疫學機制提供理論依據(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.

【學位授予單位】：中國科學技術(shù)大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R392

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