未成熟树突状细胞的体外培养及生物免疫学功能研究

发布时间：2018-07-05 17:44

本文选题：树突状细胞 + 体外培养　；参考：《第一军医大学》2005年硕士论文

【摘要】：目的: 建立一种简便实用的体外培养未成熟树突状细胞的方法,并鉴定和研究其形态、表型以及生物免疫学功能。观察不同成熟状态的树突状细胞对T淋巴细胞功能的影响,探讨未成熟树突状细胞在诱导机体产生免疫耐受中的作用,为树突状细胞在降低移植免疫排斥反应和治疗某些自身免疫性疾病的临床应用提供一些理论和实验依据。方法:1.小鼠骨髓源树突状细胞的培养:取小鼠的骨髓细胞作为树突状细胞的前体细胞,利用细胞因子rmGM-CSF和IL-4联合刺激,手法筛选树突状细胞集落,培养6天后收集疏松贴壁细胞,即为小鼠骨髓来源的树突状细胞。将培养的树突状细胞分成两组,一组在培养结束前18h加入LPS刺激其成熟(以下称成熟DC组);一组不加入LPS刺激,使其保留未成熟树突状从不特性(以下称未成熟DC组)。 2.树突状细胞的形态学观察:在光镜、电镜下观察两组细胞的形态,比较成熟组树突状细胞与未成熟组树突状细胞形态学上的差别。 3.树突状细胞的表型测定:利用流式细胞术检测两组细胞表面标志,检测小鼠树突状细胞的特异性标志CD11c评估树突状细胞纯度;检测MHC-Ⅱ类分子以及共刺激分子(CD40,CD86等)评价树突状细胞成熟度。 4.树突状细胞分泌IL-12的水平测定:收集两组树突状细胞培养上清,用ELISA法检测上清中IL-12的表达水平,了解树突状细胞分泌IL-12的水平与其成
[Abstract]:Objective:
To establish a simple and practical method for the cultivation of immature dendritic cells in vitro, and to identify and study its morphological, phenotypic and Bioimmunological functions. To observe the effect of dendritic cells in different mature states on the function of T lymphocytes, and to explore the role of immature dendritic cells in inducing immune tolerance in the body, which is a dendritic shape. The cells provide some theoretical and experimental evidence for reducing the transplant rejection and treating some autoimmune diseases.
Methods: 1. mouse bone marrow derived dendritic cells were cultured: the mouse bone marrow cells were taken as the precursor cells of the dendritic cells. The dendritic cells were selected by the combination of cytokine rmGM-CSF and IL-4 to screen the dendritic cells and collect the loose adherent cells for 6 days, that is, the dendritic cells from the bone marrow of the mice. Two groups were divided into two groups. One group was added to LPS to stimulate maturity before the end of the culture (hereinafter referred to as the mature DC group); a group did not add LPS stimulation to retain the unmature dendritic uncharacteristic (hereinafter referred to as the immature DC group).
2. morphological observation of dendritic cells: the morphology of two groups of cells was observed under light microscope and electron microscope, and the morphological difference between dendritic cells from mature group and immature group was compared.
3. the phenotypic determination of dendritic cells: detection of two groups of cell surface markers by flow cytometry, detection of specific markers of dendritic cells in mice by CD11c, to evaluate the purity of dendritic cells, and to evaluate the maturity of dendritic cells by detecting MHC- class II molecules and co stimulators (CD40, CD86, etc.).
4. the level of IL-12 secreted by dendritic cells: collecting two groups of dendritic cells culture supernatant, using ELISA to detect the expression level of IL-12 in the supernatant, to understand the level of IL-12 in the dendritic cells and its formation.
【学位授予单位】：第一军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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