左旋咪唑体外调节树突状细胞诱导Th1型免疫应答的分子机制初探

发布时间：2018-03-07 23:30

  本文选题：树突状细胞　切入点：左旋咪唑　出处：《内蒙古大学》2014年硕士论文　论文类型：学位论文

【摘要】：左旋咪唑(LMS)作为佐剂,能够诱导抗原呈递细胞产生炎性因子和上调共刺激分子CD80和CD86的表达,但对于左旋咪唑如何加强Thl细胞免疫的细胞学基础及其分子机制仍未确定。那么左旋咪唑作佐剂是否通过TLRs介导的信号通路调控树突状细胞的成熟,从而产生诱导Thl型细胞因子的微环境,从而增强Th1型免疫应答,到目前尚未见此方面的相关研究报道。因此,本项目拟对左旋咪唑佐剂在提高机体细胞免疫尤其是Thl型细胞免疫应答的机理作一探索。 以DC表面的TLRs为靶位,研究左旋咪唑佐剂与DC相互作用的效应,并在此基础上探讨二者相互作用的受体和信号转导机制,旨为佐剂作用的机理研究和新型疫苗研制和应用提供新的资料。 首先使用骨髓来源细胞因子(GM-CSF、IL-4)诱生法培养树突状细胞,LMS刺激细胞后,采用光学显微镜观察法、流式细胞术及ELISA法,分别观察细胞形态、检测细胞表面分子及细胞因子,对树突状细胞进行鉴定；其次采用MTT法检测LMS刺激成熟的树突状细胞诱导脾脏Naive T细胞活化增殖效应,并通过ELISA法检测树突状细胞、T细胞共培养上清中IFN-y、IL-4的分泌量来初步确定T细胞分化方向；最后通过Real-time PCR、Western blotting检测LMS刺激成熟树突状细胞表面TLR2、4、6、9,并且通过抑制剂试验进一步确定TLRs,之后Real-time PCR、Western blotting检测信号通路中下游信号接头分子MyD88及核转录因子NF-B来确定LMS刺激DC成熟信号通路。 结果如下：1、体外成功培养出小鼠骨髓来源树突状细胞前体细胞,在LMS刺激下可大量表达表面特异性分子(CD11C、MHC-Ⅱ)和共刺激分子(CD80和86)并且高表达细胞因子IL-12p70,由此证明左旋咪唑可以在体外诱导DC成熟。2、体外试验证实LMS刺激成熟的DC可以诱导Naive T细胞活化增殖,ELISA检测活化的T细胞大量分泌IFN-Y而IL-4的分泌量基本无变化,初步表明成熟的DC诱导Naive T细胞向Th1型细胞分化。3、Real-time PCR、Western blotting检测LMS刺激成熟DC高表达TLR2,anti-TLR2处理DC前体细胞后再使用LMS刺激IL-12p70表达量显著下降,由此证明LMS刺激未成熟DC成熟有TLR2的参与。4、左旋咪唑通过TLR2激活树突状细胞成熟后,信号通路接头分子MyD88及转录因子NF-B的表达均上调,由此证明LMS刺激未成熟DC成熟有TLR2介导的信号通路的参与。本研究将为进一步揭示左旋咪唑刺激未成熟树突状细胞成熟机理提供理论指导。
[Abstract]:As an adjuvant, levamisole can induce the production of inflammatory factors and up-regulate the expression of CD80 and CD86 in antigen-presenting cells. However, it is not clear how levamisole enhances the cellular immunity of Thl cells and its molecular mechanism. So whether levamisole acts as an adjuvant to regulate the maturation of dendritic cells through the signaling pathway mediated by TLRs. Therefore, the microenvironment of inducing Thl cytokines and enhancing the immune response of Th1 type have not been reported. This project aims to explore the mechanism of levamisole adjuvant in enhancing cellular immune response, especially Thl type. The effects of levamisole adjuvant on DC surface TLRs were studied, and the receptor and signal transduction mechanism of the interaction between DC and levamisole adjuvant were discussed. The aim is to provide new information for the study of the mechanism of adjuvant action and the development and application of new vaccines. Dendritic cells were first stimulated by bone marrow derived cytokine (GM-CSF- IL-4). The morphology of cells was observed by optical microscopy, flow cytometry and ELISA, respectively, and cell surface molecules and cytokines were detected. MTT assay was used to detect the effect of LMS on the activation and proliferation of Naive T cells induced by mature dendritic cells. In order to determine the differentiation direction of T cells, the secretion of IFN-ytr IL-4 in the supernatant of dendritic cells was detected by ELISA method. Finally, Real-time PCR Western blotting was used to detect the TLR2O4 / TLR9 on the surface of mature dendritic cells stimulated by LMS, and the TLRs were further determined by inhibitor test. Then the Real-time PCR Western blotting was used to detect the downstream signal junction molecule MyD88 and the nuclear transcription factor NF-B in the signaling pathway to determine LMS stings. Excitation DC mature signal pathway. The results were as follows: 1. Mouse bone marrow-derived dendritic cell progenitor cells were successfully cultured in vitro. The expression of surface specific molecules (CD11CnMHC- 鈪，

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