RelB基因沉默对骨髓树突状细胞生物免疫学功能的影响研究
发布时间：2018-08-18 11:54
【摘要】：目的： 树突状细胞(dendritic cell，DC)具有激活免疫应答或诱导免疫耐受的功能，未成熟状态的树突状细胞倾向于诱导免疫耐受产生，被称为“致耐受DC(tolerogenic dendritic cell，Tol DC)”。我们在成功建立体外扩增高纯度小鼠骨髓树突状细胞(bone marrow-derived dendritic cell，BM-DC)方法的基础上，利用最新的RNA干扰技术(RNA interference，RNAi)针对DC成熟的NFκB依赖途径中关键基因—RelB基因，筛选出最佳的siRNA PCR表达框序列，用于构建RelB基因沉默的RNAi RelB DC，并从细胞形态、细胞表面分子表达、细胞免疫功能等方面对RNAi RelB DC的生物免疫学特性进行了比较研究，以期构建出RelB基因沉默的全新致耐受DC-RNAi RelB DC，验证其诱导T细胞免疫耐受特性及可能机制，为其成功用于治疗移植排斥反应及自身免疫性疾病提供可靠的理论和实验依据。 方法： 1、骨髓DC的体外培养与鉴定研究： 分离C57BL／6小鼠骨髓前体细胞，在重组小鼠粒细胞-巨噬细胞集落刺激因子(rmGM-CSF)和重组白细胞介素-4(rmIL-4)刺激下，手法筛选并结合CD11c~+免疫磁珠分选方法，，培养扩增骨髓来源DC(BM-DC)，并通过细菌脂多糖(LPS)刺激诱导DC成熟。实验分成两组：①Control-DC组：手法筛选后免疫磁珠分选的第6天的DC细胞，即未成熟DC组；②LPS-DC组：在培养结
[Abstract]:Objective: dendritic cells (dendritic cells) have the function of activating immune response or inducing immune tolerance. Immature dendritic cells tend to induce immune tolerance, known as "tolerant DC (tolerogenic dendritic cells Tol DC". On the basis of successfully establishing a method to amplify high purity mouse bone marrow dendritic cells (bone marrow-derived dendritic BM-DC) in vitro, we used the latest RNA interference technique (RNA interference RNAi) to target the key gene -RelB gene in the mature NF 魏 B dependent pathway of DC. The best siRNA PCR expression frame sequence was selected to construct the RNAi RelB DC, silenced by RelB gene and the bioimmunological characteristics of RNAi RelB DC were compared from the aspects of cell morphology, cell surface molecular expression, cellular immune function and so on. The aim of this study was to construct a novel tolerant DC-RNAi RelB DCS induced by RelB gene silencing, to verify its characteristic and possible mechanism of inducing T cell immune tolerance, and to provide a reliable theoretical and experimental basis for the successful treatment of allograft rejection and autoimmune diseases. Methods: 1. In vitro culture and identification of bone marrow DC: Bone marrow progenitor cells of C57BL/6 mice were isolated. Bone marrow-derived DC (BM-DC) was cultured and amplified under the stimulation of recombinant granulocyte-macrophage colony stimulating factor (rmGM-CSF) and recombinant interleukin-4 (rmIL-4). DC maturation was induced by bacterial lipopolysaccharide (LPS) stimulation. The experiment was divided into two groups: 1: 1 Control-DC group: after manual screening, DC cells were sorted by immunomagnetic beads on the 6th day, namely immature DC group: 2LPS-DC group: in culture knot
【学位授予单位】：第一军医大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392.11
本文编号：2189394
[Abstract]:Objective: dendritic cells (dendritic cells) have the function of activating immune response or inducing immune tolerance. Immature dendritic cells tend to induce immune tolerance, known as "tolerant DC (tolerogenic dendritic cells Tol DC". On the basis of successfully establishing a method to amplify high purity mouse bone marrow dendritic cells (bone marrow-derived dendritic BM-DC) in vitro, we used the latest RNA interference technique (RNA interference RNAi) to target the key gene -RelB gene in the mature NF 魏 B dependent pathway of DC. The best siRNA PCR expression frame sequence was selected to construct the RNAi RelB DC, silenced by RelB gene and the bioimmunological characteristics of RNAi RelB DC were compared from the aspects of cell morphology, cell surface molecular expression, cellular immune function and so on. The aim of this study was to construct a novel tolerant DC-RNAi RelB DCS induced by RelB gene silencing, to verify its characteristic and possible mechanism of inducing T cell immune tolerance, and to provide a reliable theoretical and experimental basis for the successful treatment of allograft rejection and autoimmune diseases. Methods: 1. In vitro culture and identification of bone marrow DC: Bone marrow progenitor cells of C57BL/6 mice were isolated. Bone marrow-derived DC (BM-DC) was cultured and amplified under the stimulation of recombinant granulocyte-macrophage colony stimulating factor (rmGM-CSF) and recombinant interleukin-4 (rmIL-4). DC maturation was induced by bacterial lipopolysaccharide (LPS) stimulation. The experiment was divided into two groups: 1: 1 Control-DC group: after manual screening, DC cells were sorted by immunomagnetic beads on the 6th day, namely immature DC group: 2LPS-DC group: in culture knot
【学位授予单位】：第一军医大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392.11
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