巨噬细胞中表皮生长因子受体激活对细胞因子及实验性结肠炎的调节作用
发布时间：2019-06-11 00:29
【摘要】：巨噬细胞是先天免疫系统的重要组成部分,在免疫调节中充当重要角色。很多研究从巨噬细胞活化及调节细胞因子分泌、迁移等角度,研究肠道炎症的发病机理。表皮生长因子(EGF, epidermal growth factor)信号通路激活后可促进细胞生长,但是巨噬细胞中EGF信号通路的作用尚未阐明。本课题组一直从事肠道炎症中EGF信号通路作用的基础研究。本课题即在前期研究的基础上,探索巨噬细胞中EGF受体(EGFR, epidermal growth factor receptor)激活对细胞因子及肠道炎症的调节作用。 本课题研究分为以下三个部分： 第一部分,首先在体外实验中证明了在巨噬细胞中抑制EGFR激酶活性能够上调脂多糖(LPS, lipopolysaccharide)和干扰素-γ(IFN-γ,interferon-γ)刺激下的炎性细胞因子,如肿瘤坏死因子(TNF, tumor necrosis factor)和白介素-10(IL-10)的表达,其与p38及核因子-κB (NF-κB)表达增加相关。其次在巨噬细胞EGFR表达缺陷小鼠模型中,发现巨噬细胞中敲除EGFR能够上调LPS刺激的炎性细胞因子表达。 第二部分,利用葡聚糖硫酸钠(DSS, dextran sulfate sodium)诱发的小鼠结肠炎模型,发现巨噬细胞中敲除EGFR能够促进结肠炎缓解并增强组织修复。在炎症急性期和修复期中,巨噬细胞中敲除EGFR后对TNF-α和IL-10具有不同的调节作用。随后验证了在巨噬细胞EGFR表达缺陷小鼠模型中,IL-10参与介导了EGFR对巨噬细胞免疫应答的调节作用,巨噬细胞EGFR表达缺陷小鼠与对照组相比,IL-10表达水平在结肠炎急性期及恢复期均持续增加,而TNF表达水平在急性期增加,在恢复期减少,Anti-IL-10中和抗体能够消除上述作用。可见IL-10能够抑制TNF生成,从而对巨噬细胞EGFR表达缺陷小鼠DSS结肠炎起到保护作用。 第三部分,在巨噬细胞EGFR表达缺陷小鼠DSS结肠炎中,发现结肠上皮细胞增殖增加,组织中ki67阳性细胞增加,同时结肠巨噬细胞中及结肠组织中环氧合酶-2(cyclooxygenase-2, COX-2) mRNA水平增加。利用免疫荧光法检测溃疡性结肠炎(ulcerative colitis, UC)患者结肠镜活检标本,发现EGFR磷酸化水平及人巨噬细胞标志物CD68表达水平增加。 综上所述,可以得出如下结论：在巨噬细胞中敲除EGFR后能够增加致炎细胞因子TNF及抗炎细胞因子IL-10的生成,IL-10生成增加会抑制TNF的生成,从而对肠道炎症起到保护作用。
[Abstract]:Macrophages are an important part of innate immune system and play an important role in immune regulation. Many studies have studied the pathogenesis of intestinal inflammation from the point of view of macrophage activation and regulation of cytokine secretion and migration. The activation of epidermal growth factor (EGF, epidermal growth factor) signaling pathway can promote cell growth, but the role of EGF signaling pathway in macrophages has not been clarified. Our research group has been engaged in the basic research of EGF signaling pathway in intestinal inflammation. On the basis of previous research, this study explored the regulatory effect of EGF receptor (EGFR, epidermal growth factor receptor) activation on cytokines and intestinal inflammation in macrophages. This study is divided into the following three parts: in the first part, it was proved in vitro that inhibition of EGFR kinase activity in macrophages can up-regulate lipopolysaccharide (LPS, lipopolysaccharide) and interferon-gamma (IFN- gamma). The expression of inflammatory cytokines stimulated by interferon- 纬, such as tumor necrosis factor (TNF, tumor necrosis factor) and IL-10 (IL-10), was correlated with the increased expression of p38 and nuclear factor-kappa B (NF- kappa B). Secondly, in macrophage EGFR expression deficient mouse model, it was found that knockout EGFR in macrophages could up-regulate the expression of LPS-stimulated inflammatory cytokines. In the second part, using the mouse colitis model induced by dextran sodium sulfate (DSS, dextran sulfate sodium), it was found that knockout of EGFR in macrophages could promote the remission of colitis and enhance tissue repair. In the acute phase and repair stage of inflammation, the knockout of EGFR in macrophages has different regulatory effects on TNF- 伪 and IL-10. Then it was verified that IL-10 was involved in the regulation of macrophage immune response by EGFR in macrophage EGFR expression deficient mouse model. Compared with the control group, macrophage EGFR expression deficient mice were involved in the regulation of macrophage immune response. The expression of IL-10 continued to increase in both acute and convalescent colitis, while the expression of TNF increased in acute phase and decreased in convalescent phase. Anti-IL-10 neutralizing antibody could eliminate the above effects. It can be seen that IL-10 can inhibit TNF production, thus protecting DSS colitis in mice with macrophage EGFR expression deficiency. In the third part, in DSS colitis of mice with macrophage EGFR expression deficiency, it was found that the proliferation of colon epithelial cells increased, ki67 positive cells increased, and cycloxygenase-2 (cyclooxygenase-2,) was found in colon macrophages and colon tissues. COX-2) mRNA level increased. Colonoscopy biopsies from patients with ulcerative colitis (ulcerative colitis, UC) were detected by immunofluorescence. It was found that the phosphorylation of EGFR and the expression of CD68, a human macrophage marker, were increased. In conclusion, the following conclusions can be drawn: knockout of EGFR in macrophages can increase the production of inflammatory cytokine TNF and anti-inflammatory cytokine IL-10, and the increase of IL-10 production will inhibit the production of TNF. Thus, it plays a protective role in intestinal inflammation.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R574.62
本文编号：2496850
[Abstract]:Macrophages are an important part of innate immune system and play an important role in immune regulation. Many studies have studied the pathogenesis of intestinal inflammation from the point of view of macrophage activation and regulation of cytokine secretion and migration. The activation of epidermal growth factor (EGF, epidermal growth factor) signaling pathway can promote cell growth, but the role of EGF signaling pathway in macrophages has not been clarified. Our research group has been engaged in the basic research of EGF signaling pathway in intestinal inflammation. On the basis of previous research, this study explored the regulatory effect of EGF receptor (EGFR, epidermal growth factor receptor) activation on cytokines and intestinal inflammation in macrophages. This study is divided into the following three parts: in the first part, it was proved in vitro that inhibition of EGFR kinase activity in macrophages can up-regulate lipopolysaccharide (LPS, lipopolysaccharide) and interferon-gamma (IFN- gamma). The expression of inflammatory cytokines stimulated by interferon- 纬, such as tumor necrosis factor (TNF, tumor necrosis factor) and IL-10 (IL-10), was correlated with the increased expression of p38 and nuclear factor-kappa B (NF- kappa B). Secondly, in macrophage EGFR expression deficient mouse model, it was found that knockout EGFR in macrophages could up-regulate the expression of LPS-stimulated inflammatory cytokines. In the second part, using the mouse colitis model induced by dextran sodium sulfate (DSS, dextran sulfate sodium), it was found that knockout of EGFR in macrophages could promote the remission of colitis and enhance tissue repair. In the acute phase and repair stage of inflammation, the knockout of EGFR in macrophages has different regulatory effects on TNF- 伪 and IL-10. Then it was verified that IL-10 was involved in the regulation of macrophage immune response by EGFR in macrophage EGFR expression deficient mouse model. Compared with the control group, macrophage EGFR expression deficient mice were involved in the regulation of macrophage immune response. The expression of IL-10 continued to increase in both acute and convalescent colitis, while the expression of TNF increased in acute phase and decreased in convalescent phase. Anti-IL-10 neutralizing antibody could eliminate the above effects. It can be seen that IL-10 can inhibit TNF production, thus protecting DSS colitis in mice with macrophage EGFR expression deficiency. In the third part, in DSS colitis of mice with macrophage EGFR expression deficiency, it was found that the proliferation of colon epithelial cells increased, ki67 positive cells increased, and cycloxygenase-2 (cyclooxygenase-2,) was found in colon macrophages and colon tissues. COX-2) mRNA level increased. Colonoscopy biopsies from patients with ulcerative colitis (ulcerative colitis, UC) were detected by immunofluorescence. It was found that the phosphorylation of EGFR and the expression of CD68, a human macrophage marker, were increased. In conclusion, the following conclusions can be drawn: knockout of EGFR in macrophages can increase the production of inflammatory cytokine TNF and anti-inflammatory cytokine IL-10, and the increase of IL-10 production will inhibit the production of TNF. Thus, it plays a protective role in intestinal inflammation.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R574.62
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4 Avi Levin;Oren Shibolet;;Toll-like receptors in inflammatory bowel disease-stepping into uncharted territory[J];World Journal of Gastroenterology;2008年33期
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