摘要
支气管哮喘是危及全人类的一种严重疾病,其病因和发病机制目前尚未完全清楚。在治疗上非常棘手,只能控制,不能完全根除。免疫学机制研究表明哮喘患者存在Th1/Th2亚群比例失衡和功能失衡,Th2占优势,主要表现为Th2细胞的数量增多和功能亢进,是哮喘的始动机制和核心。巨噬细胞是免疫系统重要的细胞,有提呈抗原、清除异物及分泌多种细胞因子等作用。在通过共刺激分子和细胞因子信号途径激活和调节T细胞中发挥着独特的作用。一定条件下,能诱导Th1/Th2型应答的偏移,参与哮喘的发生发展。本实验主要定位于巨噬细胞,研究其对淋巴细胞分化的影响,以及临床常用的治疗哮喘药物茶碱对巨噬细胞的调节作用。
目的:(1)在重组人粒-巨噬细胞集落刺激因子(rhGM-CSF)作用下体外诱导培养人外周血单核源性巨噬细胞,并进行鉴定和功能检测。(2)探讨巨噬细胞对哮喘儿童Th细胞亚群(Th1/Th2)功能的影响,以及茶碱的调节作用。
方法:(1)贴壁法分离人外周血单核细胞,用含10%胎牛血清的RPMI1640培养基和rhGM-CSF培养7天。光镜、扫描电镜及透射电镜观察细胞形态,鸡红细胞吞噬试验检测吞噬功能,流式细胞术检测单核巨噬细胞表面特征性标志CD14等生物学技术鉴定贴壁细胞性质。(2)培养哮喘儿童单核源性巨噬细胞。分两组:抗原提呈组和茶碱干预组,茶碱干预组在培养过程中加入氨茶碱。两组巨噬细胞体外抗原活化后再与自体淋巴细胞共同培养3天,另设不参与反应的自身淋巴细胞对照组。流式细胞仪检测3组CD4+T细胞胞内细胞因子IFN-γ和IL-4水平,RT-PCR检测淋巴细胞转录因子T-bet和GATA-3 mRNA表达。
结果:(1)获得的贴壁细胞具备巨噬细胞的形态学特征及免疫表型,有吞噬功能,纯度较高。(2)与自身对照组和抗原提呈组相比,茶碱干预组淋巴细胞胞内IL-4水平和GATA-3 mRNA表达显著降低(P<0.05),IFN-γ/IL-4和T-bet/GATA-3比例明显升高(P<0.05)。抗原提呈组与自身对照组相比,各指标无统计学差异。3组间IFN-γ水平和T-bet mRNA表达无显著改变。
结论:(1)人外周血单核细胞在rhGM-CSF诱导下向巨噬细胞分化,具有生物学活性,纯度较高,是一种简单易行的体外诱导培养巨噬细胞的方法。(2)茶碱可通过调节哮喘巨噬细胞的功能,下调淋巴细胞GATA-3 mRNA表达,抑制Th2分泌IL-4,从而抑制Th2功能以改善哮喘Th1/Th2失衡状态。
Asthma is a serious global disease which etiological factor and pathogenesis are uncertain. The effective therapy has not been found yet, and the clinical effect is not satisfied in all patients. Immunological researches have showed that the balance of Th1/Th2 is disordered, and the exaggerated Th2-biased immune response plays a crucial role in the onset and development of asthma. Macrophages are important cells in immunological system, having many functions such as presenting antigen, eliminating foreign body and secreting various cytokines, etc. They also have notable effects on activating and regulating T cells by co-stimulatory molecules and cytokines signal pathway. Under some specific condition, macrophages can efficiently induce the migration of Th1/Th2. In this experiment, we mainly study the effects of monocyte-derived macrophages on intracellular cytokines expression of Th1/Th2 in asthmatic children and the modulatory effects of theophylline.
Objective: (1) To induce and cultivate human monocyte-derived macrophages with rhGM-CSF in vitro .The cells was identified and the biological function was examined. (2) To observe the effects of monocyte-derived macrophages on intracellular cytokines expression of Th1/Th2 in asthmatic children and the modulatory effects of theophylline.
Method: (1) Mononuclear cells isolated from human peripheral blood were incubated in RPMI1640 medium supplemented with 10% heat-inactivated FBS in the presence of rhGM-CSF for 7 days. The adherent cells were estimated to be macrophage according to morphology, phagocytosis of chicken erythrocytes and the expression of cell surface antigen. (2) The monocyte-derived macrophages of asthmatic children were cultured with or without aminophylline. After stimulated with antigen, macrophages and lymphocytes were cocultured for 3 days. The intracellular expressions of IFN-γand IL-4 in CD4+T cell were measured by flowcytometry, and the mRNA expressions of T-bet and GATA-3 were detected by RT-PCR.
Result: (1)The adherent cells displayed the morphological characteristic and immunophenotype of macrophage, had biological function.(2) In contrast with own control group and antigen presentation group, the level of intracellular IL-4 and the expression of GATA-3 mRNA in lymphocytes decreased significantly(P<0.05), and the ratio of IFN-γ/IL-4 and T-bet/GATA-3 increased significantly(P<0.05) in aminophylline added group. There was no significant difference between own control group and antigen presentation group.
Conclusion: (1) This method was simple and feasible for induction, cultivation and identification of human monocyte-derived macrophage. (2) Theophylline can effectively down regulation the mRNA expression of GATA-3, inhibit the secretion of IL-4, and regulate the balance of Th1/Th2 in asthmatic children. This effect may be mediated by macrophages.
引文
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