肺脏基质细胞对树突状细胞生物学特性的影响

发布时间：2018-06-09 15:21

本文选题：肺脏基质细胞 + 成熟树突状细胞　；参考：《泰山医学院》2010年硕士论文

【摘要】：树突状细胞(dendritic cells, DC),是体内已知功能最强的专职抗原递呈细胞(antigen presenting cells, APC),是免疫应答的中心环节,不仅能活化初始T细胞启动免疫应答,还能诱导免疫耐受负向调节免疫,有双重调节免疫反应维持免疫平衡的作用。DC的最终免疫效应取决于其发育阶段及所处的免疫微环境。免疫微环境不仅为免疫应答提供反应场所,还通过调控免疫细胞的分化发育和功能来调节免疫应答。肺脏通过气道与外界相通,每天都要接触大量外界抗原却能维持免疫平衡,可见肺脏有精确的免疫调控机制,我们通过实验证实了肺脏基质细胞微环境对免疫细胞DC的分化发育及功能有调节作用,这与维持免疫平衡有密切关系。目的观察小鼠肺脏基质细胞微环境对骨髓来源成熟树突状细胞分化发育及功能的影响,并探讨其影响机制。方法通过小鼠肺脏组织原代培养建立一株成纤维样基质细胞系,并从细胞形态及波形蛋白表达上给予鉴定,认为原代培养的肺脏基质细胞具备成纤维样细胞的特点。通过RT-PCR方法检测肺脏基质细胞高分泌两种抑制性细胞因子：TGF-β、VEGF。此基质细胞培养上清与完全培养基以1：l的比例混合后用于培养成熟树突状细胞,一周后成熟树突状细胞被诱导成具有独特生物学特性的调节性树突状细胞(DCreg)。HE染色后观察细胞形态特征,特异性夹心酶联免疫法检测培养上清细胞因子(IL-10和IL-12p70)的含量,流式细胞术检测细胞表型表达水平及吞噬能力,MTT法检测DCreg诱导同种异体T增殖能力。将上述检测结果与成熟树突状细胞进行比较,观察诱导前后树突状细胞的区别。结果调节性树突状细胞(DCreg)分泌的细胞因子IL-10高于成熟树突状细胞组(P＜0.01),而IL-12p70的分泌水平低于此组(P＜0．01)；细胞表型与成熟树突状细胞比较,CD11b上调(P0.01),CD11c、Ⅰa、CD86下调(P＜0.01)；Dcreg的吞噬能力较未成熟DC低(P＜0.01),与成熟DC无显著差异(P＞0.05)；而其诱导同种异体T增殖能力明显低于成熟DC组(mDC)。结论我们首次证实了肺脏基质细胞微环境可诱导成熟树突状细胞分化发育为另一种生物学特性不同的DC亚群--调节性树突状细胞(DCreg),其有抑制同种异体T细胞增殖进而诱导免疫耐受负向调节免疫的作用。明确了解肺脏的免疫调节机制对预防和治疗呼吸系统的过敏性疾病有很大的帮助。
[Abstract]:Dendritic cells (DC) are the most powerful professional antigen presenting cells known in vivo. They are the central link of the immune response. They not only activate the initial T cells to initiate the immune response, but also induce the immune tolerance to negative regulatory immunity. The final immune effect of DC depends on its developmental stage and its immune microenvironment. Immune microenvironment not only provides a response site for immune response, but also regulates immune response by regulating the differentiation, development and function of immune cells. The lungs communicate with the outside world through the airway. They are exposed to a large number of external antigens every day, but they can maintain the immune balance. It can be seen that the lungs have precise immunomodulation mechanisms. We have confirmed that the microenvironment of pulmonary stromal cells can regulate the differentiation, development and function of immune cells. Objective to observe the effects of microenvironment of mouse lung stromal cells on the differentiation, development and function of mature dendritic cells derived from bone marrow. Methods A fibroblast-like stromal cell line was established by primary culture of mouse lung tissue and identified from cell morphology and vimentin expression. It is believed that the primary cultured pulmonary stromal cells have the characteristics of fibroblast. RT-PCR was used to detect the hypersecretion of two inhibitory cytokines: TGF- 尾 and VEGF in lung stromal cells. The culture supernatant of the stromal cells was mixed with a complete medium of 1: l and used for the culture of mature dendritic cells. After one week, mature dendritic cells were induced into regulatory dendritic cells with unique biological characteristics. The morphological characteristics of the cells were observed after staining, and the levels of IL-10 and IL-12p70 in the supernatants were detected by specific sandwich enzyme-linked immunosorbent assay (Elisa). The phenotypic expression and phagocytosis of T cells were detected by flow cytometry. MTT assay was used to detect the ability of DCreg to induce allogeneic T proliferation. The results were compared with mature dendritic cells. Results IL-10 secreted by regulatory dendritic cells was higher than that of mature dendritic cells (P < 0.01), but the secretion level of IL-12p70 was lower than that of mature dendritic cells (P < 0.01). The phagocytic ability of CD11b was lower than that of immature DC (P < 0.01), and there was no significant difference between CD11b and mature DC (P > 0.05), but its ability to induce proliferation of allogeneic T was significantly lower than that of mature DC. Conclusion it is the first time that we have confirmed that the ability of lung stroma to proliferate is significantly lower than that of mature DC. Cellular microenvironment can induce the differentiation and development of mature dendritic cells into another kind of DC subsets with different biological characteristics-regulatory dendritic cells, which can inhibit the proliferation of allogeneic T cells and induce immune tolerance to negative regulatory immunity. A clear understanding of the immunomodulatory mechanism of the lung is of great help in the prevention and treatment of allergic diseases of the respiratory system.
【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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