维生素D受体(VDR)基因多态性通过调节单核/巨噬细胞功能参与创伤性骨髓炎发病的机制研究

In recent years, the number of patients diagnosed of post-traumatic osteomyelitis following open fractures and infection after fracture fixation following closed fractures is increasing. Nowadays, post-traumatic osteomyelitis remains to be one of the most challenging disorders for physicians. Pathogenesis of this disorder is associated with environmental and host factors. However, current investigations are still limited regarding how host hereditary susceptibility involves in the pathogenesis of this disease. Outcomes of our preliminary study revealed that vitamin D receptor (VDR) gene polymorphism ApaI site may be linked to an increased risk of developing this disease in Chinese population. Therefore, the current program is designed from perspective of single nucleotide polymorphisms (SNPs). We plan to conduct a large sample size case-control study to confirm which VDR SNP site significantly associates with the susceptibility to post-traumatic osteomyelitis, and population with which genotype may be in a higher risk to develop this disease in Chinese population. In addition, we will compare differences of VDR expressions in monocytes-macrophages and its potential influences among different genotypes of the significant SNP site. Moreover, in vitro animal and cell experiments will be performed to explore that in circumstance of inflammation, whether VDR SNP participants in the pathogenesis of post-traumatic osteomyelitis via its regulation on expressions of related serum inflammatory biomarkers, cathelicidin, phagocytosis and bacterial killing ability of the monocytes-macrophages. This program will provide explicit evidence for the role of host hereditary susceptibility in the pathogenesis of post-traumatic osteomyelitis and also, will lay the foundation for clinical screening of a higher risk population with necessary interventions.

近年来，开放性骨折伤后及闭合性骨折内固定术后感染所致创伤性骨髓炎的患者日趋增多，该病目前仍为骨科医生所面临最具挑战性的疾病之一，其发病受外界环境与宿主因素的共同作用。但迄今为止，有关宿主遗传易感性是如何影响其发病的机制研究仍然较少。我们前期预实验结果表明：维生素D受体(VDR)基因多态性位点ApaI可能显著增加国人该病的发病风险。因此，本课题立足于VDR基因多态性，通过大样本病例-对照研究，明确与国人创伤性骨髓炎发病存在显著相关性的位点及高危人群基因型；针对具有显著意义的位点，比较其不同基因型宿主外周血单核/巨噬细胞中VDR表达的差异及影响；并通过动物和细胞实验，探讨炎症环境下，VDR多态性是否通过影响单核/巨噬细胞中相关炎性因子及抗菌肽表达、吞噬作用与杀菌效力等功能而参与创伤性骨髓炎的发病，进而明确宿主遗传易感性在创伤性骨髓炎发病中的作用，为临床筛检高危人群并进行干预奠定基础。

近年来，开放性骨折伤后及闭合性骨折内固定术后感染所致创伤性骨髓炎的患者日趋增多，该病目前仍为骨科医生所面临最具挑战性的疾病之一，其发病受外界环境与宿主因素的共同作用。但迄今为止，有关宿主遗传易感性是如何影响其发病的机制研究仍然较少。本项目“聚焦前沿，独辟蹊径”，从维生素D受体(VDR)基因多态性角度揭示其在骨感染发病中的作用。首先，通过704例临床病例对照研究发现：VDR的ApaI位点与创伤性骨髓炎的发病风险存在显著相关性，突变型纯合子AA是具有保护性的基因表型；随后，比较了ApaI位点不同基因型炎性因子、LL-37及VD的表达水平，结果表明：AA表型TNF-α及SAA水平显著低于、而VD水平高于AC及CC表型，因此，后续实验以ApaI位点显著影响VD的表达水平为切入点，重点探讨了其对单核/巨噬细胞吞噬杀菌及凋亡等功能的影响。在吞噬杀菌部分，体外实验结果表明：VD可促进巨噬细胞中VDR蛋白的表达，VDR高表达可以增强巨噬细胞的吞噬杀菌能力，其可能的部分机制是通过抑制NF-κB-p65的核转位来实现。在凋亡实验部分，研究发现：金葡菌刺激能够激活NF-κB磷酸化活化，上调促凋亡信号Fas、Bax表达，下调抑凋亡信号Bcl-2表达，激活Caspase-3诱导凋亡途径；而VD能够通过提高VDR表达水平显著抑制金葡菌介导的巨噬细胞凋亡进程，VDR高表达同时激活其下游抑凋亡信号A20表达上调，二者协同抑制NF-κB的活化并抑制TNF-α表达，以此共同发挥抑凋亡效应。体外动物实验结果亦表明：VD在下调机体炎症水平的同时，抑制了金葡菌介导的单核/巨噬细胞凋亡。通过本项目实施，明确了参与创伤性骨髓炎发病的遗传学因素，为临床进行高危人群筛检并提前进行干预，以期降低骨感染的发病率奠定了基础，同时，明确了VD在骨感染治疗中的作用及其机制，为后期用于骨感染的辅助治疗，以期提高其治愈率提供了理论依据。