大肠杆菌麦芽糖结合蛋白调节肿瘤相关巨噬细胞由M2型向M1型极化作用机制研究

发布时间：2018-03-02 15:20

  本文选题：麦芽糖结合蛋白　切入点：RAW264.7细胞　出处：《吉林大学》2015年博士论文　论文类型：学位论文

【摘要】：麦芽糖结合蛋白（MBP）是大肠杆菌麦芽糖转运系统的重要成员，主要负责麦芽糖的摄取及分解代谢。以往研究认为MBP具有极低生物活性，故在分子生物学中常用作标签蛋白或作为融合蛋白用于亚单位疫苗制备。然而近期研究报道，MBP能够提高亚单位疫苗的免疫原性，并且MBP能够通过Toll样受体4（TLR4）活化树突状细胞（DC），促进细胞因子分泌。本课题组研究发现MBP作为融合蛋白能够提高MUC1肿瘤蛋白疫苗的免疫原性，MBP作为佐剂与卡介苗（BCG）联合抗肿瘤能够显著抑制小鼠Lewis肺癌以及B16黑色素瘤移植瘤的生长，增强抗肿瘤细胞免疫应答；此外，MBP能够非特异性促进小鼠脾脏淋巴细胞增殖，诱导Th1细胞活化，并且能够促进小鼠腹腔巨噬细胞分泌IL-1、IL-6和NO，增强其吞噬活性，亦可促进NK细胞活化。以上研究表明，MBP具有增强多种免疫细胞活性、诱导细胞免疫应答的作用，据此我们推测MBP可能对细胞免疫应答中的巨噬细胞具有活化作用，可能诱导巨噬细胞向M1型极化。近年研究表明，巨噬细胞在肿瘤的发生发展中具有重要的作用，不同极化类型的巨噬细胞对肿瘤的发展具有不同的作用，抑制肿瘤相关巨噬细胞（TAM）的M2型极化被认为是极具应用前景的抗肿瘤进展与转移的新策略，而研究MBP在TAM极化表型转化中的调控作用将为开发以TAM为中心的肿瘤免疫治疗策略提供一个新的方向。 本研究首先针对MBP对小鼠巨噬细胞系RAW264.7细胞的极化作用及机制进行深入探讨。通过体外细胞实验，研究MBP对RAW264.7细胞的极化作用，我们通过检测MBP诱导RAW264.7细胞NO分泌水平，细胞因子IL-1β、IL-6及IL-12p70的分泌水平，以及巨噬细胞表面分子CD80、MHC II、iNOS的表达，研究MBP诱导RAW264.7细胞的极化类型，阐明MBP能够诱导巨噬细胞向M1型极化。而后，以MBP对RAW264.7细胞TLR2与TLR4表达水平的调控为切入点，通过研究下游NF-κB和MAPK信号传导通路，深入研究MBP发挥作用的可能途径，从而揭示MBP诱导RAW264.7细胞极化的可能作用机制。进一步对MBP在TAM极化表型转化中调控作用的研究将以建立TAM体外实验模型为基础，采用肿瘤细胞培养上清（TCS）与RAW264.7细胞混合培养的方式建立体外TAM模型，以小鼠乳腺癌细胞系4T1肿瘤细胞培养上清模拟肿瘤微环境诱导RAW264.7细胞形成TAM。通过M1型与M2型巨噬细胞特征性细胞因子IL-10、IL-12p70分泌水平、表面分子iNOS、CD16/32与CD206表达的检测，判断肿瘤细胞培养上清诱导的RAW264.7细胞的极化类型，证实肿瘤细胞培养上清将诱导RAW264.7细胞向M2型极化。最后，研究MBP在TAM极化表型转化中的调控作用，，通过研究MBP对TCS诱导的TAM表型转化的影响，判断MBP是否具有调节TAM由M2型向M1型极化的作用，以M1型巨噬细胞特征性细胞因子IL-12分泌水平及分子标志iNOS与CD16/32的表达，M2型巨噬细胞特征性细胞因子IL-10的分泌水平及分子标志CD206表达水平的变化为判断依据，从而阐明MBP能够调节TAM由M2型向M1型极化。本研究不仅揭示MBP可以直接诱导巨噬细胞M1型极化，更揭示MBP具有调节TAM由M2型向M1型的翻转发挥，为MBP作为一种新型免疫增强剂的开发提供理论基础，并将为开发以TAM为中心的治疗策略提供一个新的方向。
[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″瀷涓哄熀纭

本文编号：1557064