本文選題：麥芽糖結(jié)合蛋白　切入點(diǎn)：RAW264.7細(xì)胞　出處：《吉林大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：麥芽糖結(jié)合蛋白（MBP）是大腸桿菌麥芽糖轉(zhuǎn)運(yùn)系統(tǒng)的重要成員，主要負(fù)責(zé)麥芽糖的攝取及分解代謝。以往研究認(rèn)為MBP具有極低生物活性，故在分子生物學(xué)中常用作標(biāo)簽蛋白或作為融合蛋白用于亞單位疫苗制備。然而近期研究報道，MBP能夠提高亞單位疫苗的免疫原性，并且MBP能夠通過Toll樣受體4（TLR4）活化樹突狀細(xì)胞（DC），促進(jìn)細(xì)胞因子分泌。本課題組研究發(fā)現(xiàn)MBP作為融合蛋白能夠提高M(jìn)UC1腫瘤蛋白疫苗的免疫原性，MBP作為佐劑與卡介苗（BCG）聯(lián)合抗腫瘤能夠顯著抑制小鼠Lewis肺癌以及B16黑色素瘤移植瘤的生長，增強(qiáng)抗腫瘤細(xì)胞免疫應(yīng)答；此外，MBP能夠非特異性促進(jìn)小鼠脾臟淋巴細(xì)胞增殖，誘導(dǎo)Th1細(xì)胞活化，并且能夠促進(jìn)小鼠腹腔巨噬細(xì)胞分泌IL-1、IL-6和NO，增強(qiáng)其吞噬活性，亦可促進(jìn)NK細(xì)胞活化。以上研究表明，MBP具有增強(qiáng)多種免疫細(xì)胞活性、誘導(dǎo)細(xì)胞免疫應(yīng)答的作用，據(jù)此我們推測MBP可能對細(xì)胞免疫應(yīng)答中的巨噬細(xì)胞具有活化作用，可能誘導(dǎo)巨噬細(xì)胞向M1型極化。近年研究表明，巨噬細(xì)胞在腫瘤的發(fā)生發(fā)展中具有重要的作用，不同極化類型的巨噬細(xì)胞對腫瘤的發(fā)展具有不同的作用，抑制腫瘤相關(guān)巨噬細(xì)胞（TAM）的M2型極化被認(rèn)為是極具應(yīng)用前景的抗腫瘤進(jìn)展與轉(zhuǎn)移的新策略，而研究MBP在TAM極化表型轉(zhuǎn)化中的調(diào)控作用將為開發(fā)以TAM為中心的腫瘤免疫治療策略提供一個新的方向。 本研究首先針對MBP對小鼠巨噬細(xì)胞系RAW264.7細(xì)胞的極化作用及機(jī)制進(jìn)行深入探討。通過體外細(xì)胞實(shí)驗(yàn)，研究MBP對RAW264.7細(xì)胞的極化作用，我們通過檢測MBP誘導(dǎo)RAW264.7細(xì)胞NO分泌水平，細(xì)胞因子IL-1β、IL-6及IL-12p70的分泌水平，以及巨噬細(xì)胞表面分子CD80、MHC II、iNOS的表達(dá)，研究MBP誘導(dǎo)RAW264.7細(xì)胞的極化類型，闡明MBP能夠誘導(dǎo)巨噬細(xì)胞向M1型極化。而后，以MBP對RAW264.7細(xì)胞TLR2與TLR4表達(dá)水平的調(diào)控為切入點(diǎn)，通過研究下游NF-κB和MAPK信號傳導(dǎo)通路，深入研究MBP發(fā)揮作用的可能途徑，從而揭示MBP誘導(dǎo)RAW264.7細(xì)胞極化的可能作用機(jī)制。進(jìn)一步對MBP在TAM極化表型轉(zhuǎn)化中調(diào)控作用的研究將以建立TAM體外實(shí)驗(yàn)?zāi)Ｐ蜑榛A(chǔ)，采用腫瘤細(xì)胞培養(yǎng)上清（TCS）與RAW264.7細(xì)胞混合培養(yǎng)的方式建立體外TAM模型，以小鼠乳腺癌細(xì)胞系4T1腫瘤細(xì)胞培養(yǎng)上清模擬腫瘤微環(huán)境誘導(dǎo)RAW264.7細(xì)胞形成TAM。通過M1型與M2型巨噬細(xì)胞特征性細(xì)胞因子IL-10、IL-12p70分泌水平、表面分子iNOS、CD16/32與CD206表達(dá)的檢測，判斷腫瘤細(xì)胞培養(yǎng)上清誘導(dǎo)的RAW264.7細(xì)胞的極化類型，證實(shí)腫瘤細(xì)胞培養(yǎng)上清將誘導(dǎo)RAW264.7細(xì)胞向M2型極化。最后，研究MBP在TAM極化表型轉(zhuǎn)化中的調(diào)控作用，，通過研究MBP對TCS誘導(dǎo)的TAM表型轉(zhuǎn)化的影響，判斷MBP是否具有調(diào)節(jié)TAM由M2型向M1型極化的作用，以M1型巨噬細(xì)胞特征性細(xì)胞因子IL-12分泌水平及分子標(biāo)志iNOS與CD16/32的表達(dá)，M2型巨噬細(xì)胞特征性細(xì)胞因子IL-10的分泌水平及分子標(biāo)志CD206表達(dá)水平的變化為判斷依據(jù)，從而闡明MBP能夠調(diào)節(jié)TAM由M2型向M1型極化。本研究不僅揭示MBP可以直接誘導(dǎo)巨噬細(xì)胞M1型極化，更揭示MBP具有調(diào)節(jié)TAM由M2型向M1型的翻轉(zhuǎn)發(fā)揮，為MBP作為一種新型免疫增強(qiáng)劑的開發(fā)提供理論基礎(chǔ)，并將為開發(fā)以TAM為中心的治療策略提供一個新的方向。
[Abstract]:Maltose binding protein (MBP) is an important member of the Escherichia coli maltose transport system, is mainly responsible for the maltose uptake and catabolism. Previous studies suggest that MBP has a very low biological activity, it is commonly used in molecular biology as a label or as a fusion protein for protein subunit vaccine preparation. However, recent Research Report, MBP to improve the immunogenicity of the subunit vaccine, and MBP through Toll like receptor 4 (TLR4) activation of dendritic cells (DC), promote the secretion of cytokines. The research group found that MBP as a fusion protein can enhance the immunogenicity of tumor MUC1 protein vaccine and MBP as adjuvant with BCG (BCG) combined with anti tumor can inhibit the growth of mouse Lewis lung cancer and B16 melanoma xenografts, enhance anti-tumor cellular immune response; in addition, MBP can promote non-specific proliferation of mouse spleen lymphocytes, induced The activation of Th1 cells, and can promote the secretion of mouse peritoneal macrophages IL-1, IL-6 and NO, enhanced the phagocytic activity, can promote the activation of NK cells. These studies show that MBP can enhance the immune cell activity, induce cell immune responses, we speculate MBP may have a role in activating the cellular immune response in macrophages that may induce macrophage polarization to M1. Recent studies indicate that macrophages play an important role in tumor development, has not the same role in the development of different types of macrophages on polarization of tumor, inhibition of tumor associated macrophages (TAM) of the M2 polarization is considered to be a new strategy for progression and metastasis potential resistance the tumor, and study the role of MBP in regulation of TAM polarization in phenotypic transformation will provide a new way for tumor immunotherapy strategies developed with TAM as the center To.
In this study, the polarization effect and mechanism of MBP on mouse macrophage RAW264.7 cells are discussed. Through the experiments in vitro, the polarization effect of MBP on RAW264.7 cells, we detected MBP RAW264.7 cells induced NO secretion, cytokine secretion and IL-6 IL-1 beta, IL-12p70, and macrophage surface molecules CD80, MHC the expression of iNOS, II, polarization type study of MBP induced RAW264.7 cells, can induce macrophage polarization to clarify MBP M1. Then, based on MBP TLR2 and TLR4 RAW264.7 expression level regulation as a starting point, through the study of the downstream NF- kappa B and MAPK signal transduction pathway, possible ways to effect the in-depth study of MBP play. In order to reveal the possible mechanism of MBP induced RAW264.7 cell polarization. Further study on the regulation of MBP in TAM polarization phenotypic effect will to establish TAM in vitro 妯″瀷涓哄熀紜

本文編號：1557064