钙卫蛋白对调节性T细胞免疫功能的影响及其对创伤后脓毒症的预警机制研究

中文摘要

英文摘要

In our previous study, we have proved that calprotectin (S100A8/S100A9), a late inflammatory cytokine described recently, was a potential immunostimulatory signal. When used at some concertration, calprotectin could effect the immune function of regulatory T cells(Tregs) in vitro. However, fundamental questions regarding the receptor mechanisms of calprotectin and Tregs remain unresolved. Toll-like receptor-4(TLR4) plays an important role in the expressing process of Treg immune function and is potentially associated with the signal regulation of calprotectin,s inflammation effect. We suspect wether TLR4 could participate the regulating process of Treg immune function mediated by calprotectin. The current study was conducted to clarify the effects of continued stimulation with calprotectin on Treg and also the intrinsic relation with TLR4 at the whole, cello- and molecular levels. We also plan to take advantage of the TLR4 antibody, shRNA disturbing technique and gene knockout mice to inhibit the activation and gene expression of TLR4, and to assess the relationships among calprotectin, Treg and TLR4 both in vitro and in vivo. On base of these experiments, we will analysis the dependabilities of calprotectin and the serious degrees of septic animals as well as septic patients after trauma, to investigate the calprotectin,s early warning mechanism to sepsis. Our findings will help to clarify the underlying effect of calprotectin on the disorder of cellular immune functions and its receptor mechanism in sepsis, thus shed a light to the development of novel therapeutic strategies for improving the clinical outcome of patients with severe injury and subsequent sepsis.

结题摘要

钙卫蛋白是一种新发现的晚期炎症介质。我们前期研究发现，一定浓度的钙卫蛋白体外刺激对调节性T细胞(Treg)免疫功能具有调节作用，但其确切受体机制尚不清楚。Toll样受体4(TLR4)在钙卫蛋白致炎作用的信号调控及Treg免疫功能发挥过程中均具重要作用，因此推测其可能参与了钙卫蛋白介导Treg免疫功能过程。本项目从整体、细胞和分子水平观察了钙卫蛋白对Treg免疫功能及TLR4活化的影响，并利用TLR4抗体、shRNA质粒及KO小鼠在体内/外抑制TLR4活化和基因表达，探讨了钙卫蛋白对Treg免疫功能影响的变化及其与TLR4表达水平的内在联系。实验结果提示，钙卫蛋白确实可在体内外通过TLR4途径，以下调Treg细胞表面抑制性分子的表达及抑制性细胞因子的分泌两条途径下调Treg的功能。在此基础上，通过临床试验，分析了钙卫蛋白与创伤后脓毒症患者病情严重程度的相关性，结果发现：（1）外科大手术术后1、2、3、5、7 d，脓毒症组患者的血清钙卫蛋白水平分别为（5.6±0.8）、（7.7±1.2）、（9.6±1.5）、（7.4±1.1）、（5.0±0.5）μg/mL均显著高于非脓毒症组[（3.3±0.4）、（4.1±0.6）、（4.7±0.8）、（4.0±0.5）、（3.5±0.5）μg/mL，t值分别为1.735~4.304，P<0.05或P<0.01]。（2）外科术后1-7 d，脓毒症组患者外周血的IL-2、IL-6、IFN-γ、CRP水平、PCT水平、动脉血乳酸水平、APACHE Ⅱ评分和SOFA评分均显著高于非脓毒症组。（3）经单因素及多因素Logistic回归分析显示，确诊患者血清钙卫蛋白水平（优势比为12.7，95%置信区间为6.7～22.3，P<0.01）及PCT（优势比为15.4，95%置信区间为8.8～26.5，P<0.01）是影响患者并发脓毒症的独立危险因素。（4）ΔSC1-3及ΔPCT1-3（确诊脓毒症后第3d与第1 d差值）的ROC曲线下总面积分别为0.86、0.88；最佳阈值分别为2.5μg/mL、3.7ng/mL；二者对脓毒症预测的敏感度分别为87%、89%，特异度分别为89%、90%。本研究从全新的角度进一步认识了钙卫蛋白在介导脓毒症细胞免疫功能紊乱中的具体作用和相关受体机制，为探索脓毒症的免疫调控途径提供了新的思路。