轻、重度脊柱结核Th1-OC骨免疫轴差异表达及1 ,25 (OH) 2 D3调控作用机制研究

轻、重度脊柱结核骨质破坏特点、预后截然不同，其OC功能表达差异化机制不清。课题组发现VD缺乏抑制抗结核功能，Tb激活的Th1诱导OC过程中，INF-γ通过RANKL-RANK信号通路抑制OC功能。推测：VD缺乏导致脊柱结核微环境中OC过表达、骨破坏重，VD对Tb-Th1-OC骨免疫轴有浓度依赖性调控作用。本项目检测轻、重度脊柱结核患者病灶细胞因子差异化表达，FISH比较OC内共定位、功能基因定量检测；VD干预轻、重度脊柱结核患者OC，qPCR、WB定量检测OC分化、融合功能相关基因、蛋白表达；进一步建立VD缺乏、VDR KO和VD正常小鼠Th1-OC共培养体系，生理浓度VD干预，并增强或抑制IFN-γ信号通路，qPCR、WB定量检测共培养体系中骨免疫相关基因与蛋白表达，定性检测OC分化、融合及功能，深入研究3个信号通路传话影响，以阐明脊柱结核骨破坏差异性原因，为防治骨关节结核奠定基础。

Chemotherapy only management is recommended for mild spinal tuberculosis, while surgery should be considered for severe spinal tuberculosis with major complications. The characteristics of bone destruction in mild and severe spinal tuberculosis were pretty different, and these led to different prognosis. The major issue is that the mechanisms of differential expression of osteoclast in these still remains unclear. Our previous studies found that VD deficiency inhibited the immune function against TB in mice, the anti-IFN-γ antibody induced osteoclast formation, and is probably mediated by RANKL induced activation of NF-κB, that induces TRAF6 in the RANKL-RANK signaling pathway. We hypothesize that VD deficiency may lead to over expression of OC in the microenvironment of spinal tuberculosis and more bone resorption, the 1, 25 (OH) 2 D3 may affect the Tb-Th1-OC osteoimmunology in a concentration dependent manner. In this study, these are divided into mild spinal tuberculosis group, severe spinal tuberculosis group, and control group. we are going to test the level of cytokine in the focus of mild and severe spinal tuberculosis by ELISA, the immune associated genes are analyzed by microarray. The co-location of RANKL and TRAF6 inside the OC is observed by FISH. Isolated culture of human osteoclast cells from patients with mild and severe spinal tuberculosis are performed, then the OC are treated with 1, 25 (OH) 2 D3 of biological concentration. The biomarkers, specific genes, fusogenic genes and functional genes in each differentiation stage during osteoclastogenesis were examined by qPCR. The fusogenic and functional related proteins expression were tested by Western blot. In the further study, the mouse are divided into Vitamin D deficiency group, VDR knockout group and normal group. Tuberculosis activiated Th1 cells and OC are co-cultured with gradient concentrations of 1,25(OH)2D3 using transwell, the influence and mechanism of 1,25(OH)2D3 on Th1 cell mediated osteoclastogenesis are evaluated using qPCR,western blot, immunohistochemistry, TRAP stain and CFA stain as above. Retest all above after strengthening or blocking the IFN-γ signa lpathway.Those are expected to resolve the dispute and reduce tuberculosis incidence and develop modulating immunity as a potential therapy for osteoarticular tuberculosis.