表没食子儿茶素没食子酸酯对髓源性巨噬细胞极化的影响

发布时间：2018-04-29 07:07

本文选题：骨髓细胞 + 表没食子儿茶素-3-没食子酸酯　；参考：《河南师范大学》2017年硕士论文

【摘要】：巨噬细胞是一类具有高度异质性和可塑性的免疫细胞群体,起源于骨髓的单核细胞,能够改变自己的表型以适应于所处微环境。巨噬细胞极化分为经典活化型巨噬细胞(classically activated macrophage,M1)和替代活化型巨噬细胞(alternativelly activated macrophage,M2)两种类型。巨噬细胞在γ-干扰素(IFN-γ)、细菌脂多糖(LPS)刺激下分化为M1型巨噬细胞,分泌多种促炎细胞因子、活性氧等,如TNF-α、IL-1β、ROS等以清除病原体、坏死组织和激活其他免疫细胞,帮助机体抵御感染。M2型巨噬细胞由IL-4、IL-13诱导产生,可分泌多种抗炎因子,如Ym-1、IL-10等,主要出现在炎症消退阶段有助于组织修复。表没食子儿茶素没食子酸酯(epigallocatechin-3-gallate,EGCG)是绿茶多酚的主要活性成分,具有抗氧化、抗炎、抗癌等多种生物学效应。EGCG抗癌和抗炎作用机制已有较多的研究。但EGCG能否影响髓源性巨噬细胞的极化,目前并不清楚。因此,本实验通过观察EGCG对髓源性巨噬细胞分化的M1型巨噬细胞和M2型巨噬细胞的标志性因子的表达情况探讨EGCG对髓源性巨噬细胞极化的影响。本实验以6~8周龄C57BL/6小鼠骨髓细胞为材料,经100 ng/mL巨噬细胞集落刺激因子(macrophage colony-stimulating factor,M-CSF)体外诱导分化为巨噬细胞(bone marrow-derived macrophages,BMDM),加入50 ng/mL的IFN-γ和1μg/m L的LPS刺激分化为M1型巨噬细胞,或者加入20 ng/mL的IL-4刺激分化为M2型巨噬细胞,然后暴露于12.5~50μmol/L的EGCG处理,7天后收集细胞提取RNA和蛋白,利用实时荧光定量PCR(RT-PCR)和酶联免疫吸附试验(ELISA),检测M1型巨噬细胞标志物i NOS和促炎因子IL-1β、TNF-α以及M2型巨噬细胞标志物Arg-1、Ym-1和抗炎因子IL-10的mRNA表达水平和蛋白含量。结果显示,IFN-γ和LPS刺激使髓源性巨噬细胞的IL-1β、TNF-α和iNOS mRNA和蛋白水平上调,而EGCG处理可抑制IFN-γ和LPS刺激的IL-1β、TNF-α和iNOS表达,且具有剂量依赖效应。IL-4刺激使髓源性巨噬细胞的Arg-1、Ym-1、IL-10表达上调,EGCG处理组可增强Arg-1、Ym-1、IL-10的表达,且存在剂量依赖效应。上述结果表明,EGCG以剂量依赖方式减弱M1型巨噬细胞标志因子的表达,而增强M2型巨噬细胞标志因子的表达。
[Abstract]:Macrophages are a class of immune cells with high heterogeneity and plasticity. They are derived from bone marrow mononuclear cells and can change their phenotypes to adapt to their microenvironment. The polarization of macrophages can be divided into two types: classical activated activated macrophage M _ 1) and alternative activated activated macrophage M _ 2). Macrophages, stimulated by IFN- 纬, lipopolysaccharide (LPS), differentiate into M1 macrophages, secrete a variety of pro-inflammatory cytokines and reactive oxygen species, such as TNF- 伪, IL-1 尾 Ros, in order to clear pathogens, necrotic tissue and activate other immune cells. The macrophages can be induced by IL-4 and IL-13, and secrete many kinds of anti-inflammatory factors, such as Ym-1, IL-10 and so on. Epigallocatechin-3-gallate EGCGG is the main active component of green tea polyphenols, and has many biological effects, such as anti-oxidation, anti-inflammation, anti-cancer and so on. The mechanism of anti-cancer and anti-inflammatory effects of EGCG has been studied. However, it is not clear whether EGCG can affect the polarization of myelogenous macrophages. Therefore, the effect of EGCG on the polarization of myelogenous macrophages was investigated by observing the expression of the marked factors of M1 type macrophages and M2 type macrophages of myelogenous macrophages. In this study, bone marrow cells of C57BL/6 mice aged 6 to 8 weeks were used as materials. Macrophage colony-stimulating factor M-CSF (100 ng/mL macrophage colony-stimulating factor) was used to induce the differentiation of macrophages into marrow-derived macrophages BMDM, and 50 ng/mL IFN- 纬 and 1 渭 g / mL LPS were added to induce macrophages to differentiate into M1 macrophages. Alternatively, 20 ng/mL IL-4 was added to stimulate differentiation into M2 macrophages, and then exposed to 12.5 渭 mol/L EGCG for 7 days, the cells were collected to extract RNA and protein. The mRNA expression and protein content of M 1 macrophage markers I NOS, IL-1 尾 TNF- 伪, M 2 macrophage markers Arg-1Tm-1 and anti-inflammatory factor IL-10 were detected by real-time fluorescence quantitative PCR and Elisa assay. The results showed that IFN- 纬 and LPS increased the levels of IL-1 尾 -TNF- 伪 and iNOS mRNA and protein in myelogenous macrophages, while EGCG treatment inhibited the expression of IL-1 尾 -TNF- 伪 and iNOS stimulated by IFN- 纬 and LPS. The expression of Ym-10 in myelogenous macrophages increased in a dose-dependent manner after stimulation with IL-4. In EGCG treated group, the expression of IL-10 was enhanced in a dose-dependent manner, and there was a dose-dependent effect on the expression of IL-10. These results suggested that EGCG could attenuate the expression of M1 type macrophage marker factor in a dose-dependent manner, but enhance the expression of M 2 type macrophage marker factor.
【学位授予单位】：河南师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392

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