1,25(OH)2D3对结核激活的Th1细胞介导破骨细胞生成的影响及机制研究

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。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。