【作者】 叶丽萍；

【导师】 王春凤；

【作者基本信息】 吉林农业大学 ， 预防兽医学， 2017， 博士

【摘要】 轮状病毒（Rotavirus,RV）感染已成为全球性危害公共卫生安全的主要原因之一,RV感染机体首先入侵肠道,诱导机体免疫应答以肠道黏膜免疫为主,目前对其感染特点、免疫反应及疫苗开发做了大量的研究工作,但对其具体的黏膜免疫分子机制尚未阐明。黏膜免疫是连接固有免疫和获得性免疫的桥梁,其中树突状细胞（dendritic cells,DCs）作为重要的抗原递呈细胞起着衔接固有免疫和获得性免疫的关键免疫分子作用。本研究用猪轮状病毒（Porcine Rotavirus,PRV）体外刺激小鼠骨髓源树突状细胞（Bone marrow-derived dendritic cells,BMDCs）和体内感染BALB/c小鼠,以DCs表面的Toll样受体（Toll-like receptors,TLRs）为靶位,研究PRV刺激对DCs成熟活化、亚群分化、细胞因子分泌及Th分化等的影响;并在此基础上通过Pepinh-TRIF阻断BMDCs的TRIF信号和感染TRIF^-/-小鼠,研究PRV通过DCs诱导的黏膜免疫分子机制,为PRV疫苗开发和疾病防治奠定理论基础。PRV体外刺激GM-CSF和IL-4诱导的小鼠BMDCs,应用流式细胞术、荧光定量PCR、ELISA和Western blotting方法检测PRV刺激对BMDCs的影响结果表明:（1）PRV刺激能促进BMDCs的活化与成熟,上调表达MHC II和共刺激分子CD40、CD80和CD86,增加DCs的抗原递呈能力。（2）PRV刺激BMDCs后TLR2和TLR3（而不是TLR4和TLR8）参与PRV刺激后的免疫应答。（3）PRV刺激BMDCs活化的TLR3/TLR2可通过TRAF3或TRAF6诱导NF-κB活化和细胞因子IL-6、IL-10、IL-12、IFN-γ等的分泌。（4）PRV刺激成熟的BMDCs能显著促进T淋巴细胞增殖并促进T细胞向Th1类细胞分化。将BALB/c小鼠随机分成PBS对照组、PRV组和poly（I:C）组进行PRV攻毒试验,检测PRV刺激对小鼠脾脏和MLN中DCs成熟活化、亚群分化、细胞因子分泌影响的结果表明:（1）PRV感染可促进小鼠脾脏和MLN中DCs表面MHC II和共刺激分子CD40、CD80和CD86表达增加,并向CD11c⁺CD11b⁺CD8α⁺、CD11c⁺CD11b⁺CD8α^-、CD11c⁺B220⁺CD8α⁺和CD11c⁺B220⁺CD8α^-亚群分化。（2）PRV感染小鼠MLN和小肠中TLR2、TLR3、MyD88和NF-κB mRNA随着攻毒时间的延长表达量逐渐增加,具有时间依赖性。（3）PRV感染初期脾脏和MLN分泌细胞因子以IL-10和IL-6为主,后期以IL-12和IFN-γ的Th1型细胞因子为主。PRV体外刺激以Pepinh-TRIF预处理培养至第8 d的BMDCs,应用流式细胞术、荧光定量PCR和ELISA方法检测阻断TRIF信号PRV对BMDCs的影响结果表明:（1）阻断TRIF信号下调表达BMDCs表面MHC II和共刺激分子CD40、CD80和CD86,抑制了BMDCs的成熟与活化。（2）阻断TRIF信号下调表达TRAF3和TRAF6抑制NF-κB活化,减弱BMDCs刺激T淋巴细胞增殖的能力。（3）阻断TRIF信号对PRV刺激BMDCs分泌细胞因子具有抑制作用,减弱了PRV刺激BMDCs对T淋巴细胞分化的能力。将TRIF^-/-小鼠和C57BL/6小鼠随机分成TRIF^-/-小鼠PBS对照组、TRIF^-/-小鼠PRV组、TRIF^-/-小鼠poly（I:C）组、WT PBS对照组、WT PRV组和WT poly（I:C）组进行PRV攻毒试验,检测PRV感染TRIF基因缺失小鼠对脾脏和MLN中DCs成熟活化、亚群分化、细胞因子分泌等的实验结果表明:（1）PRV感染TRIF^-/-小鼠减弱了MLN中DCs向CD11c⁺CD11b⁺CD8α⁺DCs、CD11c⁺CD11b⁺CD8α^-DCs、CD11c⁺B220⁺CD8α⁺DCs、CD11c⁺B220⁺CD8α^-DCs亚群分化能力,对DCs的成熟与活化也有一定的抑制作用。（2）PRV感染TRIF^-/-小鼠TRAF3和NF-κB mRNA下调表达,但对MyD88和TRAF6 mRNA的影响较小。（3）PRV感染TRIF^-/-小鼠,抑制了MLN中细胞因子的分泌下调表达IL-6、IL-10、IL-12和IFN-γ,影响T细胞活化。实验研究发现PRV刺激DCs通过TLR2/MyD88信号通路和TLR3/TRIF信号通路诱导NF-κB活化,刺激IL-6、IL-10、IL-12、IFN-γ等细胞因子的分泌参与免疫应答。阻断TRIF信号或缺失TRIF基因对DCs的成熟活化具有抑制作用,对DCs介导的TRAF3/NF-κB信号通路影响较大,而对MyD88/TRAF6信号通路的影响较小。DCs介导的黏膜免疫在PRV感染的作用机制和了解PRV感染的免疫机制及有效预防PRV感染方面具有重要意义。更多还原

【Abstract】 Rotavirus（RV）infection is a global heath problem,researchers from all over the world had numerous studies on infection characteristics,immune response and vaccine development of rotavirus.Evidence is accumulating that RV infection of the body’s first invasion of intestinal epithelial cells and triggers mucosal immune responses,however,little is known about the mucosal immune mechanisms.Mucosal immune is a bridge connecting innate and adaptive immune,and dendritic cells（DCs）which are professional antigen-presenting cells（APCs）serve as the key immune molecules.We utilized PRV to stimulate mouse bone marrow-derived dendritic cells（BMDCs）and gastric BALB/c mice,explored the Toll-like receptors which is regarded as one typical PAMP on the surface of DCs.Furthermore,we explored the signal transduction mechanism in TRIF^-/-mice and BMDCs pretreated with Pepinh-TRIF in vivo.The interaction between BMDCs and PRV was investigated in vitro using flow cytometry,Quantitative real-time PCR,ELISA and western blotting analysis.The experimental results showed:（1）PRV could induce the activation and maturation of BMDCs,up-regulated the levels of MHC II and costimulatory molecules CD40,CD80 and CD86.（2）Toll-like receptor 2（TLR2）and TLR3（rather than TLR4 and TLR8）were involved in immune responses after PRV stimulation.（3）TLR2 and TLR3 got involved in the activation of NF-κB and cytokine（IL-6,IL-10,IL-12,IFN-γ）secretion by BMDCs through TRAF3 or TRAF6.（4）The PRV-stimulated BMDCs also promote the proliferation of T lymphocytes and induce the differentiation of na?ve T cells into Th1 cells.BALB/c mice were divided randomly into three groups:PBS control,PRV and poly（I:C）group.The activation,subgroup differentiation and cytokines secretion of DCs infected with PRV were investigated.The experimental results showed:（1）The expression of MHC II and costimulatory molecules（CD40,CD80 and CD86）increased in DCs of spleen and MLN.The differentiation of CD11c⁺CD11b⁺CD8α⁺,CD11c⁺CD11b⁺CD8α^-,CD11c⁺B220⁺CD8α⁺and CD11c⁺B220⁺CD8α^-was observed.（2）The mRNA levels of TLR2,TLR3,MyD88 and NF-κB in MLN and small intestine increased gradually with time elapsed after PRV infection.（3）In the early stage of viral infection,IL-10 and IL-6 secretion took up the majority in spleen and MLN,but Th1 cytokines（IL-12 and IFN-γ）were secreted mainly in late stage.The interaction between Pepinh-TRIF pretreatement BMDCs and PRV was investigated using flow cytometry,Quantitative real-time PCR and ELISA in vitro.The experimental results indicated:（1）Blocking TRIF signaling could down-regulate the levels of MHC II,CD40,CD80and CD86,then inhibited activation and maturation of BMDCs.（2）Down-regulated TRAF3 and TRAF6 could inhibit NF-κB activation,weakened the ability to induce the T lymphocytes proliferation after blocking TRIF signaling.（3）Blocking TRIF signaling could inhibit cytokines secretion of BMDCs and weak the differentiation ability of T lymphocytes by BMDCs.BALB/c and TRIF^-/-mice were divided randomly into six groups:TRIF^-/-PBS control,TRIF^-/-PRV,TRIF^-/-poly（I:C）,WT PBS control,WT PRV and WT poly（I:C）group.The activation,subgroup differentiation and cytokine secretion of DCs of TRIF deletion infected with PRV were investigated.The experimental results indicated:（1）Differentiation ablity of DCs into CD11c⁺CD11b⁺CD8α⁺DCs,CD11c⁺CD11b⁺CD8α^-DCs,CD11c⁺B220⁺CD8α⁺DCs and CD11c⁺B220⁺CD8α^-DCs was attenuated.Activation and maturation of DCs in MLN were inhibited to some certain extent.（2）Inhibited the expression levels of TRAF3 and NF-κB mRNA,while MyD88 and TRAF6 mRNA expression were hardly influenced.（3）The secretion of IL-6,IL-10,IL-12 and IFN-γwere abated in MLN of TRIF^-/-mice infected with PRV which affected the activation of T lymphocytes.The research deeply explored the mechanism for the interaction between PRV and DCs.PRV could trigger DCs to activate NF-κB through TLR2/MyD88 signaling pathway or TLR3/TRIF signaling pathways.PRV is also involved in IL-6,IL-10,IL-12 and IFN-γsecretion in the immune response.Blocking TRIF signaling or TRIF deletion could inhibit the activation and maturation of DCs stimulated with PRV,inhibited the TRAF3/NF-κB signaling pathway and hardly influenced MyD88/TRAF6 signaling pathway.Further studied on the mucosal immune mechanisms of DCs stimulated with PRV is of a great significance for understanding the PRV infection pathogenesis,then we could contrive an effective prevention against PRV infection.更多还原