耐多药脊柱结核患者MΦ-OC功能失调及1,25(OH)2D3调节机制研究

Multi-drug resistant spinal tuberculosis presents severer bone destruction, higher recurrence rate, the managements for it are pretty challenging. Due to poor efficacy of the first-line and/or second-line drugs in fighting against MDR-TB, modulating immunity with VD shows attractive potentials, but still remains controversial. Our previous studies found that VD deficiency inhibited the immune function against TB in mice, VD regulated the maturation and function of DC to affect the acquired immune.1,25(OH)2D3 affected the Tb-Th1-OC immune network in a concentration dependent manner. So we hypothesize that 1,25 (OH) 2D3 could enhance the killing effect of macrophage on MDR-TB and inhibit the he differentiation, fusion and bone resorption of osteoclast. Then we are focusing on macrophage-osteoclast dysfunction and 2 signal pathways for these. Those are divided into observing group with no VD, positive control group with gradient concentrations of 1,25(OH) 2D3, and negative control group with VDR siRNA. Immune associated genes are analyzed by microarray. The autophagy and polarization related genes and proteins expression were tested by Real Time PCR and Western Blot, retest all above afer blocking the PPARγ. 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. Through regulation of cytokine dosage and induction time, TRAP stain, CFA stain, fusion assay and pit formation assay were used to evaluate activity of osteoclasts. Those are expected to resolve the dispute, to provide further evidence of preliminary clinical trials.

耐多药脊柱结核骨质破坏重、复发率高，治疗特别困难。VD辅助免疫调节潜力诱人，但存争议。课题组发现VD缺乏抑制抗结核功能、影响DC成熟及功能，VD在TB-Th1-OC骨免疫网络的调节具有浓度依赖性。推测1,25(OH)2D3可增强MΦ对耐多药结核杀灭作用，并抑制OC分化、功能。本项目研究MDR-TB激活的MΦ-OC功能失调及2个信号通路调节机制，设不干预、VD干预、VDR siRNA沉默组，Microarray比较MΦ免疫相关基因表达，Real Time PCR和Western Blot检测自噬、极化相关基因及蛋白表达，并阻断PPARγ对比；进一步建立OC体外诱导模型，qPCR检测分化各阶段标志物、特异基因、融合基因和功能基因表达，Western blot定量检测融合、功能蛋白表达，TRAP和CFA染色、融合和噬骨试验定性检测分化、融合和功能，有望解决争议，为下一步临床RCT研究提供依据。

维生素D（VD）的经典作用是调节钙的吸收和稳态，骨代谢以及细胞的生长和分裂。1,25(OH)2D3作为具有生物活性的VD，已经被证明能够调节细胞的生长、迁移、分化和免疫应答。VD缺乏与许多病理状况有关，包括感染、自身免疫和过敏性疾病。前期课题组成功建立VD缺乏小鼠动物模型，VD缺乏显著抑制小鼠抗结核免疫功能，提示VD缺乏与结核病易感性相关，且VD缺乏者患结核后病情严重。文献报道的大剂量 VD 辅助治疗肺结核的随机、双盲临床研究发现，VD不仅能提高临床疗效，还能改善患者的影像学表现，但在骨结核中没有相关的研究报道。临床上，骨结核患者尤其是VD缺乏的患者表现出严重的骨质破坏，我们猜想可能是由于结核分枝杆菌通过TLR激活了巨噬细胞的炎症信号通路，从而促进了破骨细胞的生成，导致了骨破坏，但VD在结核引起破骨细胞异常激活中的作用尚不清楚。.在2020年有关结核的会议报告上提出了“大幅度增加对结核病的预防性治疗”的目标。在此背景下，本研究旨在探究1,25(OH)2D3与VDR在结核导致的异常破骨激活中的作用及其机制。在抗结核新药和治疗性抗结核疫苗研发无重大进展的背景下，VD的免疫调节作用具有诱人的潜力，促使我们研究在化疗基础上辅助VD免疫调节治疗耐多药结核，以期增强杀菌效果、减轻结核病变程度和提高治愈率。.1、1,25(OH)2D3在破骨细胞的形态、基因表达以及功能发挥等方面均显示出抑制作用，且具有一定的浓度依赖性，在0.1-1000nM范围内浓度越高抑制作用越显著。.2、结核分枝杆菌(M. tb)感染导致异常的破骨细胞激活。.3、1,25(OH)2D3抑制结核分枝杆菌(M. tb)诱导的异常破骨细胞生成，并具有浓度依赖性。.4、1,25(OH)2D3通过VDR抑制结核分枝杆菌(M. tb)诱导的异常破骨细胞生成。.5、VDR的活化通过抑制IκB磷酸化抑制M. tb诱导的破骨细胞形成。.研究结论.本研究表明，结核分枝杆菌感染导致破骨细胞生成过度激活，VDR显著上调。1,25(OH)2D3激活VDR可通过IκB α/p65信号通路抑制M. tb诱导的异常的破骨细胞生成（分化、融合和骨吸收活性）。随着耐多药结核病的日益严重，迫切需要新的药物治疗的开发。本研究结果揭示了1,25(OH)2D3作为一种新药在预防和改善骨关节结核方面的潜在价值。