【摘要】：目的： 樹突狀細胞(dendritic cell，DC)具有激活免疫應(yīng)答或誘導免疫耐受的功能，未成熟狀態(tài)的樹突狀細胞傾向于誘導免疫耐受產(chǎn)生，被稱為“致耐受DC(tolerogenic dendritic cell，Tol DC)”。我們在成功建立體外擴增高純度小鼠骨髓樹突狀細胞(bone marrow-derived dendritic cell，BM-DC)方法的基礎(chǔ)上，利用最新的RNA干擾技術(shù)(RNA interference，RNAi)針對DC成熟的NFκB依賴途徑中關(guān)鍵基因—RelB基因，篩選出最佳的siRNA PCR表達框序列，用于構(gòu)建RelB基因沉默的RNAi RelB DC，并從細胞形態(tài)、細胞表面分子表達、細胞免疫功能等方面對RNAi RelB DC的生物免疫學特性進行了比較研究，以期構(gòu)建出RelB基因沉默的全新致耐受DC-RNAi RelB DC，驗證其誘導T細胞免疫耐受特性及可能機制，為其成功用于治療移植排斥反應(yīng)及自身免疫性疾病提供可靠的理論和實驗依據(jù)。 方法： 1、骨髓DC的體外培養(yǎng)與鑒定研究： 分離C57BL／6小鼠骨髓前體細胞，在重組小鼠粒細胞-巨噬細胞集落刺激因子(rmGM-CSF)和重組白細胞介素-4(rmIL-4)刺激下，手法篩選并結(jié)合CD11c~+免疫磁珠分選方法，，培養(yǎng)擴增骨髓來源DC(BM-DC)，并通過細菌脂多糖(LPS)刺激誘導DC成熟。實驗分成兩組：①Control-DC組：手法篩選后免疫磁珠分選的第6天的DC細胞，即未成熟DC組；②LPS-DC組：在培養(yǎng)結(jié)
[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
【學位授予單位】：第一軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2006
【分類號】：R392.11

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