本文選題：禽流感滅活全病毒 + 鼻腔黏膜�。� 參考：《南京農(nóng)業(yè)大學》2015年博士論文

【摘要】：禽流感(Aivan Influenza,AI)已給我國和世界上許多國家的養(yǎng)禽業(yè)造成了極大的經(jīng)濟損失。禽流感主要分為高致病性和低致病性禽流感。雖然低致病禽流感的發(fā)病和死亡率均較低,但宿主可以作為攜帶者傳播病毒,為流感病毒變異重組提供了資源庫。鼻腔是禽流感病毒進入機體的主要入口之一。研究表明經(jīng)鼻腔免疫可以在局部建立有效的黏膜免疫應答,直接切斷病毒的感染路徑。然而,由于鼻腔黏膜屏障的阻礙,單獨應用滅活流感病毒進行鼻腔免疫時不能有效地提升機體免疫力。近年來,CpG ODN(CpG Oligodeoxynucleotides)佐劑因其具有較強的免疫刺激作用,已受到廣泛關(guān)注和研究。CpG ODN的主要作用機制是可以靶向并促進黏膜下樹突狀細胞(Dendritic cells,DCs)成熟,進而有效地增強黏膜和系統(tǒng)免疫應答。但是,在黏膜屏障存在的情況下,CpG ODN是否有助于禽流感滅活病毒顆粒的跨鼻腔黏膜遞送至今不明確。因此,本研究首先確證CpG ODN配合H9N2禽流感滅活全病毒(H9N2 WIV)鼻腔免疫小鼠后局部黏膜免疫和系統(tǒng)免疫應答水平。再次,在體外試驗中,CpG ODN配合H9N2 WIV直接與小鼠DCs(體外誘導培養(yǎng))相互作用,應用流式細胞術(shù)、ELISA試驗和混合淋巴細胞反應試驗,分別評估DCs的表型成熟、細胞因子表達和促進CD4~+T細胞增殖的能力。最后,在體外建立的DCs/Calu-3上皮細胞共培養(yǎng)模型和體內(nèi)小鼠鼻腔灌注模型,應用流式細胞術(shù)和共聚焦顯微鏡等方法,探討CpG誘導鼻腔黏膜下DCs跨上皮攝取H9N2 WIV并轉(zhuǎn)運至引流淋巴結(jié)-頸淋巴結(jié)的能力,同時深入研究了DCs招募和樹突形成的機制,也闡明了參與此過程的DCs亞型和捕獲受體,以期為CpG ODN作為鼻腔黏膜免疫佐劑的研究和應用提供一定的理論依據(jù)。本研究內(nèi)容分為以下三個部分:1、CpG ODN配合H9N2 WIV鼻腔免疫對小鼠呼吸道局部黏膜和全身系統(tǒng)免疫應答水平的影響本試驗應用CpG ODN配合H9N2 WIV滴鼻免疫小鼠,通過檢測鼻腔、氣管和肺各呼吸道中IgA的特異性抗體水平;血清中IgG及其亞型抗體水平;中和HI水平;脾淋巴細胞的活化(CD69表達)、增殖和亞群變化的情況,評價小鼠局部黏膜和全身系統(tǒng)的免疫應答水平。結(jié)果發(fā)現(xiàn):應用CpG ODN配合H9N2 WIV滴鼻免疫小鼠28天后,鼻腔、氣管和肺涮洗液中IgA抗體水平、血清中IgG及其亞型抗體水平、中和HI水平均顯著(p0.05)高于單獨H9N2 WIV免疫后的水平。此外,脾淋巴細胞活化和增殖能力顯著提升,CD3~+CD4~+T細胞亞群比例也有大幅上升。然而,單獨應用H9N2 WIV免疫后,以上指標均未顯著上調(diào)(p0.05).結(jié)果表明:CpG ODN作為鼻腔黏膜佐劑,配合H9N2 WIV后可以有效地提升小鼠呼吸道局部黏膜及全身的系統(tǒng)免疫應答水平。2、CpG ODN配合H9N2 WIV對小鼠樹突狀細胞活化和成熟的影響DCs是連接天然免疫和獲得性免疫的重要的紐帶。DCs活化和成熟直接影響著下游免疫應答水平。因此,本試驗首先從小鼠骨髓中分離骨髓前體細胞,并應用GM-CSF和IL-4聯(lián)合誘導前體細胞分化為DCs,并通過形態(tài)學、純度和功能進行鑒定。隨后,應用CpG ODN配合H9N2 WIV體外刺激已培養(yǎng)成功的DCs 24 h,收集DCs和上清液,分別檢測DCs的成熟表型標志和細胞因子的表達。此外,另一部分收集的DCs與異種的CD4~+T細胞混合培養(yǎng),檢測CD4~+T細胞的增殖情況。結(jié)果顯示:CpG ODN配合H9N2 WIV后,與空白對照組相比,顯著上調(diào)CD40和CD80的表達;同時,促進IL-12p70和IL-10分泌;在DCs與CD4~+ T細胞的混合淋巴細胞反應試驗中,CD4~+ T細胞顯著增殖。結(jié)果表明:CpG ODN配合H9N2 WIV后可以有效地促進DCs活化和成熟,可能是引起機體較好免疫應答水平的重要機制。3、CpG ODN對鼻腔黏膜下DCs跨上皮攝取H9N2 WIV的影響CpG ODN是否能夠有效協(xié)助H9N2 WIV跨鼻腔黏膜遞送,目前仍不清楚。在本研究中,通過體外DCs/Calu-3共培養(yǎng)模型試驗和體內(nèi)小鼠鼻腔灌注試驗,發(fā)現(xiàn)CpG ODN能有效協(xié)助H9N2 WIV招募DCs至鼻腔黏膜下區(qū)域,并伸出跨上皮樹突捕獲腔側(cè)的病毒粒子。其中,CD103~+亞型DCs參與了以上過程,SIGN-R1是鼻腔黏膜DCs上重要的攝取H9N2 WIV的受體。鼻腔上皮細胞分泌的趨化因子CCL20在DCs招募和跨上皮樹突形成的過程中發(fā)揮重要作用。攝取病毒的鼻腔黏膜DCs能夠快速的向其引流淋巴結(jié)-頸淋巴結(jié)遷移并遞呈抗原。此外,CpG ODN并沒有影響上皮細胞自身的轉(zhuǎn)運能力,即跨細胞途徑和細胞旁通路。結(jié)果表明:CpG ODN配合H9N2 WIV后可以通過誘導黏膜下DCs形成跨上皮樹突,進而有效地攝取H9N2 WIV,這可能是CpG ODN增強下游獲得性免疫應答的新機制。
[Abstract]:Aivan Influenza (AI) has caused great economic loss to poultry industry in China and many countries in the world. Avian influenza is mainly divided into high pathogenic and low pathogenic avian influenza. Although the incidence and mortality of low pathogenic avian influenza are low, the host can transmit the virus as a carrier and provide the variant recombinant of influenza virus. The nasal cavity is one of the main entrance of the avian influenza virus into the body. The study shows that the nasal cavity immunity can establish an effective mucosal immune response in the local area and directly cut off the infection path of the virus. However, the inactivation of the inactivated influenza virus for nasal immunity can not be effectively promoted because of the obstruction of the nasal mucosa barrier. In recent years, CpG ODN (CpG Oligodeoxynucleotides) adjuvant has been widely concerned and studied the main mechanism of.CpG ODN because of its strong immune stimulation effect. It can target and promote the maturation of submucosal dendritic cells (Dendritic cells, DCs), and thus effectively enhance the mucosal and systemic immune response. In the presence of the mucosal barrier, it is not clear whether CpG ODN contributes to the transmission of the cross nasal mucosa of the avian influenza inactivated virus particles. Therefore, this study first confirms the local mucosal immunity and systemic immune response level of CpG ODN combined with H9N2 avian influenza inactivated virus (H9N2 WIV) inactivated virus (H9N2 WIV) mice. Again, in vitro, CpG ODN With the interaction of H9N2 WIV directly with mouse DCs (in vitro induced culture), flow cytometry, ELISA test and mixed lymphocyte reaction test were used to evaluate the phenotypic maturation of DCs, the expression of cytokines and the ability to promote the proliferation of CD4~+T cells. Finally, the co culture model of DCs/Calu-3 epithelial cells and the mice nose in vitro were established in vitro. Cavity perfusion model, flow cytometry and confocal microscopy were used to investigate the ability of CpG to induce DCs transepithelial uptake of H9N2 WIV under nasal mucosa and transport to lymph node and cervical lymph node. The mechanism of DCs recruitment and dendrite formation was studied, and the DCs subtype and capture receptor involved in the process were also clarified, so as to be CpG OD. N provides a certain theoretical basis for the research and application of nasal mucosal immune adjuvant. The contents of this study are divided into three parts: 1, the effect of CpG ODN combined with H9N2 WIV on the local mucosal and systemic immune response in the respiratory tract of mice. The experiment was conducted by CpG ODN combined with H9N2 WIV nose immunization in mice, through the detection of the nasal cavity, The specific antibody level of IgA in the trachea and the respiratory tract, the level of IgG and its subtype antibody in the serum, the level of neutralizing HI, the activation of the spleen lymphocyte (CD69 expression), the proliferation and subgroup changes, and evaluating the immune response level of the local mucosa and systemic system of the mice. The results showed that the use of CpG ODN combined with H9N2 WIV intranasal immune mice for 28 days After that, the level of IgA antibody in the nasal cavity, trachea and lung rinse, the level of IgG and its subtype antibody in the serum, and the level of the neutralizing HI (P0.05) were higher than that of the individual H9N2 WIV. In addition, the activation and proliferation of splenic lymphocytes increased significantly, and the CD3~+CD4~+T cell subsets were also significantly increased. However, H9N2 WIV immunization was used alone. The above indexes were not significantly up-regulated (P0.05). The results showed that CpG ODN, as a nasal mucosa adjuvant, combined with H9N2 WIV, could effectively enhance the systemic immune response level.2 in the local mucosa and whole body of the respiratory tract of mice. The effect of CpG ODN and H9N2 WIV on the activation and maturation of dendritic cells in mice was linked to natural and acquired immunity. The important link,.DCs activation and maturation, directly affects the level of the downstream immune response. Therefore, this experiment first isolated the bone marrow progenitor cells from the mouse bone marrow, and used GM-CSF and IL-4 to induce the differentiation of the precursor cells into DCs, and identified by morphology, purity and function. Then, CpG ODN combined with H9N2 WIV in vitro was used to stimulate the body. The successful DCs 24 h was cultured and DCs and supernatant were collected to detect the mature phenotypic markers of DCs and the expression of cytokine. In addition, the proliferation of CD4~+T cells was detected by the mixed culture of DCs and heterogeneous CD4~+T cells. The results showed that CpG ODN combined with H9N2 WIV, compared with the blank control group, the CD40 and the CD4~+T were significantly up-regulated. At the same time, it promotes the secretion of IL-12p70 and IL-10, and in the mixed lymphocyte reaction test of DCs and CD4~+ T cells, CD4~+ T cells proliferate significantly. The results show that CpG ODN and H9N2 WIV can effectively promote DCs activation and maturation, and may be an important mechanism to induce better immune response to the body. The effect of epithelial uptake of H9N2 WIV on CpG ODN is still unclear whether CpG ODN can effectively assist the H9N2 WIV delivery of nasal mucosa. In this study, the DCs/Calu-3 co culture model test in vitro and in vivo mouse nasal perfusion test found that CpG ODN could effectively assist H9N2 WIV to recruit DCs to submucosal region of the nasal cavity and extend the trans epithelial dendritic trap. The CD103~+ subtype DCs participates in the above process, and SIGN-R1 is an important receptor for the uptake of H9N2 WIV on the nasal mucosa DCs. The chemokine CCL20 secreted by the nasal epithelial cells plays an important role in the recruitment of DCs and the formation of the trans epithelial dendrites. Lymph nodes and cervical lymph nodes migrate and present antigen. In addition, CpG ODN does not affect the transport capacity of epithelial cells themselves, that is, cross cell pathway and paracellular pathway. The results show that CpG ODN can induce DCs to form transepithelial dendrites by inducing submucosal DCs and then effectively absorb H9N2 WIV after H9N2 WIV, which may be a CpG ODN enhanced downstream acquisition. A new mechanism for the response to the immune response.

【學位授予單位】：南京農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：S852.4

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