【摘要】 探讨通过IL-10重组慢病毒载体转染小鼠未成熟树突状细胞(iDC),构建高分泌IL-10的耐受性树突状细胞的可行性。粒细胞巨噬细胞集落刺激因子(GM-CSF)和白细胞介素4(IL-4)联合诱导并经CD11c免疫磁珠分选获得小鼠骨髓iDC,倒置显微镜下观察,流式细胞术分析细胞表面分子;IL-10重组慢病毒载体转染小鼠iDC,荧光显微镜下观察GFP表达,ELISA法测定转染细胞的IL-10表达水平,并行混合淋巴细胞反应,观察其诱导T淋巴细胞增殖的情况,流式细胞术检测调节性T细胞。结果:获得大量高纯度未成熟DC,随培养时间延长表现出典型的树突状形态,流式细胞术分析显示iDC表面MHCⅡ、CD80、CD86表达水平均显著低于mDC;未成熟DC经IL-10慢病毒转染后,荧光显微镜下观察GFP蛋白高度表达,ELISA检测IL-10表达水平明显高于对照组(P<0.05),且IL-10转染iDC体外刺激T淋巴细胞增殖的能力弱于未转染iDC,但调节性T细胞比例高于后者。IL-10基因通过慢病毒载体介导,成功整合到iDC基因内,使其高表达IL-10,基因修饰后的iDC的致免疫耐受作用得到了增强。更多还原

【Abstract】 To investigate the feasibility on the construction of tolerogenic dendritic cells characterised with high IL-10 expression and induced by IL-10 recombinated lentivirus vector,the mouse bone marrow dendritic cells(DCs) were obtained by co-induction with recombinant mouse granulocyte and macrophage colony stimulating factor(GM-CSF) and interleukin-4(IL-4),and then were purified with anti-CD11c antibody-coated magnetic beads.The morphological changes of these cells were observed with light microscope and their surface molecules such as CD80,CD86 and MHCⅡ were identified with flow cytometry.The proliferation of T lymphocytes was observed by mixed lymphocyte reaction,and the numbers of regulatory T cells were detected by flow cytometry.Mouse immature DCs were then transfected by IL-10 recombinated lentivirus vector and were observed for the expression of GFP with fluorescence microscope.The level of IL-10 expression from cultural supernatants was detected by ELISA assay.It was found that a large numbers of immature DCs with high purity were cultured and they displayed the typical morphological characteristics of DCs over the cultural time.These immature DCs had notably lower expression of CD80,CD86 and MHCⅡ than that of the mature DCs and the capacity of immature DCs to stimulate proliferation of T lymphocyte in vitro was weaker than that of mature DCs.However,the proportion of regulatory T cells was higher than that of the latter.Immature DCs transfected by IL-10 recombinated lentivirus vector had high expression of GFP as observed under fluorescence microscope,and had higher expression of IL-10 than those of the other two groups detected by ELISA assay(P<0.05).It is obvious that IL-10 gene had been successfully integrated into immature dendritic cells under the mediation of lentivirus vector,so as to make high expression of IL-10 in the cells.更多还原