TLR4信号在免疫相关造血衰竭骨髓间充质细胞过度脂肪化中的作用及活体可视研究

Based on the low immunogenicity and immune modulatory function, bone marrow mesenchymal stroma cells (BM-MSCs) have been applied to treat graft-versus-host diseases. However, recent studies indicated that MSCs were able to exert immune-stimulating effect after Toll-like receptor 4 (TLR4) on their surface was stimulated. We previously reported that activation of TLR4 pathway increased expression of B cell activating factor in BM-MSCs. We also found that T lymphocyte was a major pathogen resulting in deterioration of bone marrow microenvironment during the occurrence of immune-related BM failure. Activation of T lymphocytes was able to induce BM-MSC adipogenic differentiation and to reduce their hematopoietic supportive function. However, in immune-related BM failure, the role of TLR4 signaling pathway in MSCs' over-adipogenesis remains largely unknown. In this study, we will use TLR4-/- mice to generate immune-related BM failure model, and to investigate the regulating mechanism of TLR4 during the process of BM-MSC adipocytic differentiation in cellular immune abnormity. In addition, the spatio-temporal relationship between infiltrated T cell and hematopoietic component in bone marrow will be detected by in vivo image techniques. The result of this study will clarify the pathogenic mechanism of bone marrow-restricted immune disorders and thus help to explore the new therapeutic target for immune-related BM failure.

骨髓间充质细胞(BM-MSC)的低免疫原性和免疫调节作用，已被应用于移植物抗宿主病的治疗。但是，近来研究认为，一部分MSC具有免疫激活和炎症刺激作用，与MSC的TLR4激活有关。我们发现，TLR4激活可使BM-MSC中淋巴细胞活化因子的表达增强；而活化的T淋巴细胞可诱导BM-MSC脂肪化，使其成骨成脂分化失衡，进而削弱MSC的造血支持能力，是免疫相关造血衰竭的重要致病因子。由此我们推测，TLR4及下游信号参与了细胞免疫紊乱时BM-MSC的脂肪化进程，是免疫相关造血衰竭骨髓微环境恶化的重要因素。本课题拟以TLR4-/-的基因缺陷小鼠为载体，研究细胞免疫紊乱背景下TLR4信号通路对BM-MSC成脂成骨分化的影响，并以在体可视技术在活体小鼠中连续、动态观察浸润至骨髓内的淋巴细胞与各种造血成分相互作用的时间-空间效应。研究免疫相关造血衰竭的发生机制，对探索该类疾病免疫治疗的新靶点具有重要意义。

本研究旨在结合小动物活体荧光成像技术，探究免疫相关造血衰竭时T淋巴细胞在骨髓内与各造血成分相互作用的时间-空间效应，对探究骨髓内区域免疫疾病的发生发展、探索该类疾病免疫治疗的新靶点具有重要意义。首先，我们利用辐照+淋巴细胞输注法构建免疫相关骨髓衰竭(BMF)小鼠模型，BMF小鼠骨髓内T细胞表达最高，而且CD8+ T细胞分泌INFgamma的能力显著高于TBI组，通过检测小鼠骨髓内部微环境Th1/Th2细胞因子的表达，我们发现在BMF小鼠骨髓内部微环境存在Th1/Th2分化失衡。此外，BMF小鼠骨髓MSC成脂分化的能力增强，提示免疫紊乱可促进骨髓MSC成脂分化。其次，本课题组已构建活体可视技术研究平台，通过活体双光子显微镜和活体流式细胞仪的检测，成功观察到骨髓单个核细胞移植后的早期归巢。我们进一步以这种活体直观观察方法研究了间充质细胞对白血病肿瘤细胞的增殖和迁移侵袭，以活体影像的方法证实间充质细胞可促进白血病细胞在骨髓内的定植和归巢。本课题着重于研究细胞免疫紊乱时骨髓造血微环境的变化，并以活体小动物成像技术在体可视研究淋巴细胞与骨髓内造血成分作用的时间-空间关系，符合本计划中“病理微环境对组织器官区域免疫特性的影响和分子机制”和“组织器官区域免疫特性研究的新技术与新方法”的相关研究内容，对探究骨髓内区域免疫疾病的发生机制有重要意义。