维生素D结合蛋白对脓毒症继发多器官功能损伤的作用机制研究

Sepsis is one of a main causes of death in critically ill patients. Early and accurate diagnosis and treatment have a direct impact on the survival rate for sepsis patients. Searching for new sepsis biomarkers and exploring their related mechanisms are currently a key point in the sepsis research fields. It is reported that vitamin D binding protein (VDBP) has the potential diagnostic value for sepsis; it also participates the process of organ/tissue injury in sepsis. However, the exact mechanism remains unclear. Our previous study, through the proteomics screening biomarkers in the urine and serum of patients with clinically typical sepsis, we found that urine and serum VDBP simultaneously increased and had a significant positive correlation at the occurrence of sepsis or organ dysfunction/injury. In this study, we would reproduce the pathophysiological changes of VDBP during the sepsis and MODS process and its multiple organ injury characteristics through animal experiments in order to explore VDBP and macrophage activating factor (DBP-maf) involved in tissue and organ damage which mediate expansion of the inflammatory response and induction of local tissue apoptosis. In the cellular level, we would investigate the mechanism of VDBP and DBP-maf involved in the process of macrophage-mediated inflammation and apoptosis. Through the implementation of this subject, our study would be helpful to elucidate the mechanism of VDBP in sepsis, and to provide experimental basis for future research and treatment in sepsis.

脓毒症（sepsis）是临床危重病患者常见的死因，寻找临床上有效的sepsis生物标志物并对其相关机制进行研究是目前呼吸与危重症领域的热点问题。有研究证明维生素D结合蛋白（VDBP）对sepsis具有潜在的诊断价值并参与组织的损伤应答，但具体机制尚不清楚。我们借助蛋白质组学技术发现，当发生sepsis时患者尿液和血清中的VDBP产生明显改变，且两者呈显著正相关。我们在前期工作的基础上,拟通过动物实验探讨VDBP在sepsis和多器官功能损伤过程中的病理生理变化及其对多个脏器功能的损伤特点，研究VDBP和其形成的巨噬细胞激活因子（DBP-maf）在参与组织器官损伤中介导炎症反应的扩大和诱导局部组织细胞凋亡的作用；从细胞水平研究VDBP及其形成的DBP-maf参与巨噬细胞介导的炎症和凋亡的过程。通过本课题的实施，以期进一步阐明脓毒症的发生机制并为脓毒症的治疗提供新的干预靶点。

维生素D结合蛋白（VDBP）及维生素D相关代谢物与脓毒症的预后及发生发展有关，本课题通过运用iTRAQ 质谱及生物信息学技术筛选出脓毒症患者的尿液和血清中差异表达蛋白，发现发生sepsis 时患者尿液和血清中VDBP产生明显改变，且呈显著相关。进一步结合蛋白质生物化学、细胞生物学等方法与技术，对VDBP参与调控脓毒症的分子机制进行了初步研究。首先通过盲肠结扎穿孔法（CLP）构建小鼠脓毒症模型，比较病理损伤程度，比较VDBP的器官分布，比较不同手术组间VDBP的mRNA和蛋白表达差异与凋亡蛋白caspase3的相关性。进而应用qRT-PCR和western-blot方法比较不同浓度LPS和不同诱导时间对HepG2肝细胞系合成VDBP的影响，以及与凋亡、增殖的相关性。并通过构建稳转过表达及敲减VDBP的RAW264.7巨噬细胞株，应用流式细胞术比较VDBP不同表达水平对LPS诱导巨噬细胞凋亡的影响，进一步探讨VDBP调控LPS介导的巨噬细胞凋亡相关信号通路。VDBP对脓毒症小鼠免疫功能影响的研究正在进行。结果显示:25（OH）D3、1,25（OH）2D3及VDBP水平对脓毒症的预后具有预警价值，其中25（OH）D3是独立保护性因素；VDBP与肝功能及凝血指标具有较好的相关性，可以对脓毒症患者肝功能损伤及凝血障碍早期预测。脓毒症状态下，血清及尿液VDBP水平可以间接反映肝脏合成障碍及肺脏、脾脏等组织的损伤程度。LPS可通过影响经典凋亡通路干预肝细胞合成VDBP的能力；VDBP可通过Caspase通路、MAPK通路及NF-κB通路介导LPS刺激的巨噬细胞凋亡。综合上述结果，本研究探讨了VDBP 在脓毒症及其多器官功能损伤过程中的病理生理变化及其机制，为脓毒症的治疗提供了新的线索。项目资助会议交流3篇，发表核心论文1篇，待发表SCI1篇。培养硕博士生2名，其中1名已经取得博士学位，1名在读。项目投入经费70万元，支出59.9822万元，各项支出基本与预算相符。剩余经费10.0178万元，剩余经费计划用于本项目研究后续支出。

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