本文選題：樹突狀細(xì)胞　切入點(diǎn)：左旋咪唑　出處：《內(nèi)蒙古大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：左旋咪唑(LMS)作為佐劑,能夠誘導(dǎo)抗原呈遞細(xì)胞產(chǎn)生炎性因子和上調(diào)共刺激分子CD80和CD86的表達(dá),但對(duì)于左旋咪唑如何加強(qiáng)Thl細(xì)胞免疫的細(xì)胞學(xué)基礎(chǔ)及其分子機(jī)制仍未確定。那么左旋咪唑作佐劑是否通過(guò)TLRs介導(dǎo)的信號(hào)通路調(diào)控樹突狀細(xì)胞的成熟,從而產(chǎn)生誘導(dǎo)Thl型細(xì)胞因子的微環(huán)境,從而增強(qiáng)Th1型免疫應(yīng)答,到目前尚未見此方面的相關(guān)研究報(bào)道。因此,本項(xiàng)目擬對(duì)左旋咪唑佐劑在提高機(jī)體細(xì)胞免疫尤其是Thl型細(xì)胞免疫應(yīng)答的機(jī)理作一探索。 以DC表面的TLRs為靶位,研究左旋咪唑佐劑與DC相互作用的效應(yīng),并在此基礎(chǔ)上探討二者相互作用的受體和信號(hào)轉(zhuǎn)導(dǎo)機(jī)制,旨為佐劑作用的機(jī)理研究和新型疫苗研制和應(yīng)用提供新的資料。 首先使用骨髓來(lái)源細(xì)胞因子(GM-CSF、IL-4)誘生法培養(yǎng)樹突狀細(xì)胞,LMS刺激細(xì)胞后,采用光學(xué)顯微鏡觀察法、流式細(xì)胞術(shù)及ELISA法,分別觀察細(xì)胞形態(tài)、檢測(cè)細(xì)胞表面分子及細(xì)胞因子,對(duì)樹突狀細(xì)胞進(jìn)行鑒定；其次采用MTT法檢測(cè)LMS刺激成熟的樹突狀細(xì)胞誘導(dǎo)脾臟Naive T細(xì)胞活化增殖效應(yīng),并通過(guò)ELISA法檢測(cè)樹突狀細(xì)胞、T細(xì)胞共培養(yǎng)上清中IFN-y、IL-4的分泌量來(lái)初步確定T細(xì)胞分化方向；最后通過(guò)Real-time PCR、Western blotting檢測(cè)LMS刺激成熟樹突狀細(xì)胞表面TLR2、4、6、9,并且通過(guò)抑制劑試驗(yàn)進(jìn)一步確定TLRs,之后Real-time PCR、Western blotting檢測(cè)信號(hào)通路中下游信號(hào)接頭分子MyD88及核轉(zhuǎn)錄因子NF-B來(lái)確定LMS刺激DC成熟信號(hào)通路。 結(jié)果如下：1、體外成功培養(yǎng)出小鼠骨髓來(lái)源樹突狀細(xì)胞前體細(xì)胞,在LMS刺激下可大量表達(dá)表面特異性分子(CD11C、MHC-Ⅱ)和共刺激分子(CD80和86)并且高表達(dá)細(xì)胞因子IL-12p70,由此證明左旋咪唑可以在體外誘導(dǎo)DC成熟。2、體外試驗(yàn)證實(shí)LMS刺激成熟的DC可以誘導(dǎo)Naive T細(xì)胞活化增殖,ELISA檢測(cè)活化的T細(xì)胞大量分泌IFN-Y而IL-4的分泌量基本無(wú)變化,初步表明成熟的DC誘導(dǎo)Naive T細(xì)胞向Th1型細(xì)胞分化。3、Real-time PCR、Western blotting檢測(cè)LMS刺激成熟DC高表達(dá)TLR2,anti-TLR2處理DC前體細(xì)胞后再使用LMS刺激IL-12p70表達(dá)量顯著下降,由此證明LMS刺激未成熟DC成熟有TLR2的參與。4、左旋咪唑通過(guò)TLR2激活樹突狀細(xì)胞成熟后,信號(hào)通路接頭分子MyD88及轉(zhuǎn)錄因子NF-B的表達(dá)均上調(diào),由此證明LMS刺激未成熟DC成熟有TLR2介導(dǎo)的信號(hào)通路的參與。本研究將為進(jìn)一步揭示左旋咪唑刺激未成熟樹突狀細(xì)胞成熟機(jī)理提供理論指導(dǎo)。
[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，

本文編號(hào)：1581466