既往研究证实VD缺乏与结核易感性密切相关。课题组发现VD调节小鼠DC表型和功能来影响抗结核免疫；而VD缺乏脊柱结核患者Th1细胞活化，IFN-γ分泌增多，骨组织破坏重，机制不清。推测 1,25(OH)2D3在骨关节结核TB-Th1-OC骨免疫网络中的作用具有浓度依赖性。本项目设立小鼠VD缺乏、正常、VDR KO组，PPD诱发DTH，ELISA检测淋巴细胞因子、趋化因子表达；结核激活Th1细胞，梯度浓度1,25(OH)2D3干预，Microarray比较免疫靶基因表达差异，ELISA检测IFN-γ、RANKL和TNF-α水平，RT-PCR检测IFN-γmRNA表达，ChIP检测IFN-γ启动子区乙酰化水平；将Th1与BMMs共培养，采用基因与蛋白表达定量检测、免疫组化、SEM，探讨1,25(OH)2D3对Th1细胞介导OC生成的影响及机制，为完善结核病防治、骨关节结核免疫辅助治疗奠定基础。

Several studies confirmed that Vitamin D deficiency was associated with susceptibility to tuberculosis. We found that Vitamin D modulated the acquired immune response during mycobacterial infection by affecting the maturation and function of DCs. Th1 cells activation, increased IL-2 and IFN-γ secretion, severe bone destruction were also observed in spinal tuberculosis patients with Vitamin D deficiency. So we hypothesize that the effect of 1,25(OH)2D3 on TB-Th1-OC axis in osteoimmunology is concentration dependent.The mouse are divided into Vitamin D deficiency group, normal group and VDR knockout group. BCG and PPD are used to induce delayed type hypersensitivity, then lymphokine and lymphotactin in peripheral blood are tested by ELISA. Tuberculosis activiated Th1 cells are treated with gradient concentrations of 1,25(OH)2D3, immune associated genes are analyzed by Microarray, the secretion of IFN-γ,RANKL and TNF-α are tested by ELISA, the expression of IFN-γ mRNA is evalueated by real time-PCR and ChIP. Then Th1 cells and BMMs 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 real time PCR,western blot, immunohistochemistry and SEM. These would contribute to reduce tuberculosis incidence and develop modulating immunity as a potential therapy for osteoarticular tuberculosis.

既往研究证实VD缺乏与结核易感性密切相关，发现VD调节小鼠DC表型和功能来影响抗结核免疫；本研究：（一）探讨VD对VDR - / - 小鼠和WT小鼠中BCG感染的CD4 + T细胞的直接影响，特别是研究VD对Th1细胞的影响，阐明其分子机制。VDR - / -减弱了1,25（OH）2D3对Th1细胞分化和增殖的抑制作用。1,25（OH）2D3通过JAK / STAT通路抑制Th1细胞分化和细胞因子产生。1,25（OH）2D3影响Th细胞极化，抑制Th1和增强Th2细胞发育。此外，1,25（OH）2D3抑制炎性浸润和表达。观察VD缺乏小鼠脾淋巴液中IL-2，IFN-γ和TNF-β水平的变化。发现1,25（OH）2D3通过JAK / STAT通路抑制炎症反应，抑制Th1细胞的分化和细胞因子的产生。 1,25（OH）2D3通过抑制Th1抑制炎症损伤。 (2)将BMM与分离的Th1细胞共培养，探究了抗IFN-γ抗体对细胞分化的作用。IFN-γ抗体抑制IFN-γ，RANKL，TNF-α，MCP-1和SDF-1的细胞增殖和分泌。IFN-γ抗体处理共培养的BMMs和Th1细胞后，BMMs的细胞活力受到显着的抑制。而阴性对照兔IgG不显着改变细胞活力。抗IFN-γ抗体抑制IFN-γ的产生，但是促进了RANKL，TNF-α，MCP-1和SDF-1。IFN-γ抗体降低了IFN-γ和OPG，但增加BMM中RANKL，TNF-α，NF-κB，TRAF6和ODF的表达。IFN-γ抗体增加BMM中的肌动蛋白环形成,促进了破骨细胞的分化。IFN-γ抗体促进BMM的吸收功能,诱导破骨细胞形成。探讨1,25(OH)2D3对Th1细胞介导OC生成的影响及机制，为完善结核病防治、骨关节结核免疫辅助治疗奠定基础。