Analysis of host responses to intracellular bacteria and development of cell-mediated gene therapy

1.粒细胞-巨噬细胞集落刺激因子（GM-CSF）诱导的人单核细胞源性巨噬细胞（GM-Mφ）和巨噬细胞CSF（M-CSF）诱导的人单核细胞源性Mφ（M-Mφ）对结核分枝杆菌的耐药性不同。为了阐明参与这些Mφ之间功能差异的分子的作用，我们使用微阵列研究了基因表达谱。在用CSF培养CD 14 ^+单核细胞后，用或不用卡介苗（BCG）培养Mφ（GM-Mφ-BCG和M-Mφ-BCG）。比较这些细胞的基因表达谱。选择在M-Mφ中高度表达的趋化因子，并评估其抗分枝杆菌活性和超氧化物产生。FN 1和FCGR 2B分别是GM-Mφ和M-Mφ中表达最高的基因。经BCG刺激后，骨桥蛋白（Osteopontin，SPP 1）、CXC趋化因子配体7（CXC chemokine ligand 7，CXCL 7）和CC趋化因子配体11（CC chemokine ligand 11，CCL 11）在M-M φ-BCG中的表达明显高于GM-Mφ-BCG。对M.用SPP 1或CXCL 7刺激GM-Mφ后，观察到结核H37 Ra。SPP 1或CXCL 7刺激的GM-Mφ的超氧化物产生水平高于未刺激的GM-Mφ。这些结果表明，SPP 1和CXCL 7都可能通过增加Mφ中活性氧中间产物的产生而在抗分枝杆菌中发挥作用，至少部分是这样。2.细胞介导的基因治疗的开发对于常规治疗耐药的TB患者，计划使用具有T-bet基因的仙台病毒载体进行树突状细胞介导的基因治疗，该基因强烈诱导T细胞的Th 1发育。目前，没有获得足够的IFN-γ产量。进一步的研究将是必要的，以执行细胞介导的基因治疗。

1.Analysis of host responses to intracellular bacteriaGranulocyte-macrophage colony-stimulating factor (GM-CSF)-induced human monocyte-derived macrophage (GM-Mφ) or macrophage CSF (M-CSF)-induced human monocyte-derived Mφ (M-Mφ) are distinct in terms of the resistance to Mycobacterium tuberculosis. To elucidate the role of molecules involved in the functional differences between these Mφs, we investigated the gene expression profiles using microarray. After culture of CD14^+ monocytes with CSFs, Mφs were cultured with or without bacillus Calmette-Guerin (BCG) (GM-Mφ-BCG and M-Mφ-BCG). The gene expression profiles from these cells were compared. Chemokines highly expressed in M-Mφs were selected and evaluated for anti-mycobacterial activity and superoxide production. FN1 and FCGR2B were the most up-regulated genes in GM-Mφ and M-Mφ, respectively. After stimulation with BCG, three chemokine genes (Osteopontin (SPP1), CXC chemokine ligand 7(CXCL7) and CC chemokine ligand 11(CCL11)) were highly expressed in M-Mφ-BCG when compared to those in GM-Mφ-BCG. A significantly increased resistance to M. tuberculosis H37Ra was observed after the stimulation of GM-Mφ with SPP1 or CXCL7. Superoxide production levels of SPP1- or CXCL7-stimulated GM-Mφs were higher than those of GM-Mφs without stimulation. These results indicate that both SPP1 and CXCL7 might have a role in the resistance against mycobacteria, at least in part, through augmenting reactive oxygen intermediate production in Mφs.2.Development of cell-mediated gene therapyTo conventional therapy-resistant TB patients, dendritic cell-mediated gene therapy was planned with Sendai virus vector having T-bet gene, which strongly induces Th1 development of T cells. At present, sufficient IFN-gamma production was not obtained. Further study will be necessary to perform cell-mediated gene therapy.