【作者】 李文华；

【导师】 杨振国；

【作者基本信息】 吉林大学 ， 基础兽医学， 2013， 硕士

【摘要】 猪链球菌是一种对养猪业危害严重的革兰氏阳性致病菌，也是重要的人兽共患病原菌。猪链球菌可引起人和多种动物的脑膜炎、败血症、关节炎、肺炎、中毒性休克样综合征和心内膜炎等疾病。TLR2在猪链球菌引起的炎症应答中起着重要的作用，一旦猪链球菌活化TLR2，可促进促炎细胞因子的表达。已有研究表明，猪链球菌感染TLR2缺失的小鼠星形胶质细胞后，促炎细胞因子的表达受到显著抑制。自噬在机体抗细胞内病原菌和病毒感染过程中发挥不可或缺的作用，并能进行抗原呈递及清除胞内病原，但自噬也能导致程序性细胞死亡（PCD）。目前的研究还发现，自噬在癌症、神经退行性变、肌病、发育和老化等方面都有可能发挥作用。此外，Toll-样受体（TLRs）和自噬在天然免疫和获得性免疫中均发挥重要作用，参与对抗病原菌及病毒感染引起的免疫应答。且已有报道证实TLRs和自噬间存在相互作用关系，如TLRs配体能诱导自噬，自噬相关蛋白也可调节TLRs信号途径。因此，研究TLR2与2型猪链球菌（SS2）诱导的细胞自噬的关系，可能为猪链球菌及某些炎性病症或免疫性疾病的防治提供一定的理论基础。本实验采用直接灌胃的方式经消化道对Balb/c小鼠灌服SS2菌液，应用ELISA方法检测SS2对小鼠肠组织细胞因子分泌的影响，荧光定量PCR方法检测SS2对肠组织细胞TLR2表达的影响，Western blot方法检测SS2对肠道细胞自噬作用的影响。结果显示，SS2可促进IL-12和IL-17A的表达，激活TLR2及其下游核转录因子NF-κB，并诱导肠组织细胞的自噬。通过TLR2抗体阻断J774A.1巨噬细胞表面TLR2，再用SS2感染细胞，采用ELISA方法检测SS2和TLR2对细胞因子分泌的影响，荧光定量PCR方法检测SS2对细胞TLR2表达的影响，Western blot方法检测SS2和TLR2对细胞自噬的影响。结果显示，SS2可促进TNF-α和IL-12的表达，激活细胞内TLR2及下游核转录因子NF-κB，并诱导细胞自噬；阻断TLR2可显著抑制SS2诱导的细胞因子的分泌和细胞自噬。综上所述，SS2能通过消化道感染小鼠，引起小鼠肠组织细胞因子、TLR2和自噬作用的改变，为以后猪链球菌感染动物模型的建立提供了理论依据；TLR2参与调控SS2诱导的细胞自噬，可能成为某些免疫性疾病、癌症或老化等疾病防治新的切入点。更多还原

【Abstract】 Streptococcus suis, an important zoonotic pathogen, is a Gram-positivepathogen that seriously harms the development of the swine industry and cause aarray of deadly infections characterized as meningitis, septicemia, arthritis,pneumonia, toxic shock-like syndrome and endocarditis. TLR2plays an importantrole in the inflammatory response caused by Streptococcus suis, which can activateTLR2and promote the expression of pro-inflammatory cytokines. Previous studieshave shown that the expression of pro-inflammatory cytokines was significantlyinhibited after the TLR2-/-mice astrocytes were stimulated by Streptococcus suis.Moreover, autophagy plays an integral role against intracellular pathogens and viral,and facilitates antigen-presenting and the clearing of intracellular pathogens.However, autophagy can also lead to programmed cell death (PCD). The currentstudies also showed that autophagy may affect the process of cancer,neurodegeneration, myopathy, growth and aging. In addition, not only Toll-likereceptors (TLRs) but also autophagy plays an important role in the innate andadapted immune, participating in the immune response against microbial infections.And it has been reported that there is interaction between TLRs and autophagy, forexample, TLRs ligands could induce autophagy, and autophagy-related proteins canalso be adjusted by TLRs signaling pathways. Therefore, we studied the relationshipbetween TLR2and Streptococcus suis type2(SS2)-induced autophagy, which mayprovide a theoretical basis for prevention and treatment of Streptococcussuis-mediated diseases and certain inflammatory disorders or autoimmune diseases.In this study, Balb/c mice were challenged by intragastric administration withSS2suspension. ELISA assay was performed to detect the expression of cytokinesecreted by the intestinal tissue cells; quantitative PCR was used to detect TLR2expression of intestinal tissue cells; and Western blot assay was carried out to detectthe expression of autophagy marker protein-LC3Ⅱ. The results indicated that SS2could up-regulate the expression of IL-12and IL-17A, activate TLR2and itsdownstream nuclear transcription factor NF-kappaB, and induce autophagy of the intestinal tissue cells.J774A.1macrophage cells were infected with SS2after blocking the TLR2, akind of surface receptors of host cell. The secretion of cytokine was detected byELISA; the transcriptional levels of TLR2and NF-kappaB mRNA were analysisedby quantitative PCR; and the expression of LC3Ⅱwas determined by Western blotassay. The results suggested that SS2can up-regulate the expression of TNF-alphaand IL-12, activate intracellular TLR2and its downstream nuclear transcriptionfactor (NF-kappaB) and induce autophagy; and blocking of the TLR2couldsignificantly inhibited the secretion of SS2-induced cytokine and autophagy.Collectively, SS2might infect mice through the digestive tract, then influencethe expression of cytokines, TLR2and autophagy, which provided the theoreticalbasis for development of animal model of Streptococcus suis-mediated infections;TLR2was involved in the regulation of SS2-induced autophagy, which mayrepresent a new target for prevention and treatment of certain autoimmune diseases,cancer, aging or other diseases.更多还原