调控单核细胞趋化蛋白-1表达水平对脊柱结核免疫反应的影响及机制研究

Spinal tuberculosis is a serious disease with high morbidity and disability in China. It is essential, for improving clinical effect, to find out the relationship between the individial factors affecting development and progression in spinal tuberculosis. Studies show that monocyte chemoattractant protein-1 (MCP-1) participates in limiting the spread of tuberculosis in vivo and regulating the secondary immuneresponse. However, The mechanisms is incompletely understood. The study about the expression of MCP-1 regulating the immune response has not been reported. Our recent researchsuggestes that the distributions frequency of MCP-1 genepolymorphism in population with spinal tuberculous are different from that are normal or that in populations with lung tuberculosis. Further more,the secretion levels on MCP-1 in spinal tuberculosis patients serum are higer than others'. In this experiment, on the basis of our preliminary rusults, We plan to regulate the secretion level of MCP-1, using RNA interference technology and plasmid transfection technology, to observe its effect on monocyte-macrophage cell migration and colony formation. Secondly, we aim to establish spinal tuberculosis mice model with conditional expression of MCP-1 for further studing the role of MCP-1 in progression of spinal tuberculosis.This study is expected to lay foundation for further clinical and in-vivo research.

脊柱结核发病率高，致残率亦高。研究影响脊柱结核发生发展的免疫因素，对脊柱结核防治意义重大。研究表明: 单核细胞趋化蛋白-1（MCP-1）参与限制结核菌的体内播散并调节继发性免疫反应的发生，与结核病在体内的发生发展密切相关。但其调控机制尚不明确，MCP-1表达时机与表达量对脊柱结核免疫反应的影响更未见报道。我们新近研究发现：MCP-1基因多态性分布在正常人群、肺结核和脊柱结核人群中存在显著差异；脊柱结核患者血清MCP-1表达水平显著增高。本课题是在我们前期研究基础上，拟利用RNA干扰、质粒转染技术调节MCP-1的分泌水平，观察其对单核细胞趋化和激活能力的影响；并构建持续调控MCP-1表达的脊柱结核小鼠模型，进一步研究调控MCP-1对脊柱结核发生发展的影响。以期为更高阶段的在体及临床研究奠定基础。

脊柱结核是骨结核最常见的类型，致残率高，危害性大。手术清除往往不彻底，术后化疗时间长，且容易产生耐药结核菌。宿主的抗结核特异性免疫反应直接影响脊柱结核的发生和转归，对脊柱结核的防治意义重大。本研究中我们运用局部注射法成功构建了可调控MCP-1表达的脊柱结核小鼠模型。通过持续调控MCP-1在脊柱结核小鼠体内的表达水平，我们运用Micro-CT、免疫组化、Western-blot等手段观察到了脊柱结核病灶周围炎症细胞、蛋白、骨密度及免疫反应的差异。并进一步在脊柱结核人群中进行了验证和易感基因筛查。我们的结果显示MCP-1过表达组小鼠的椎体组织匀浆液可诱导更多的单核细胞迁徙。局部注射法构建的脊柱结核小鼠模型，成模时间短，而且更贴近脊柱结核患者的临床表现。MCP-1过表达组脊柱小鼠腰椎骨质破坏最为严重。虽然病灶椎的总体骨密度降低，但在病灶周围存在大量硬化骨形成，骨密度显著增高。我们在对小鼠脊柱结核病灶周围组织进行免疫组化后还发现了p65、INF-γ、TNF-α等免疫因子的增高，通过荧光标记，发现了大量单核细胞在病灶周围聚集。我们的结果提示MCP-1可能通过趋化单核巨噬细胞至结核病灶周围，启动并持续介导机体抗结核免疫反应。在临床病人中，我们也观察到了类似现象：脊柱结核术后并发症可能导致患者血清MCP-1浓度增高。MCP-1可以作为早期预判脊柱结核术后不良反应的生化指标之一。同时我们还筛查出MCP-1-2518 GG、MMP-1-1607 G、SP110 rs11679983AG等结核易感基因，探索了构建脊柱结核基因诊断平台的可能性。本研究通过细胞、动物及临床的一系列实验，分析了MCP-1在宿主抗结核免疫反应中的调控作用，筛查了下游相关蛋白，为下一步的干预治疗提供了方向和依据。