重症急性胰腺炎T细胞凋亡机制- - 参与[Ca2+]i调节的SOCE变化、机制和病生理意义

It has been demonstrated that TLR4 was involved in the pancreatitic injury and systemic inflammatory responses in severe acute pancreatitis (SAP), however, its role in the immunosuppression during SAP remains unknown. We have demonstrated the excessive apoptosis of T cells in SAP mice. And our preliminary data showed that, as compared to sham mice, the expression of TLR4 and STIM1 mRNA in T cells increased significantly in SAP animal model and cytosolic calcium concentration is higher than that in control mice. So, we postulat that TLR4 contributes to the development of immunosuppression during SAP by inducing T cells apoptosis via manipulating cytosolic calcium. Then, by using TLR4-/-、MyD88-/- and TRIF-/- mice, we designated firstly to evaluate the influences of TLR4 signaling on T cell apoptosis and immune function during SAP. Then, we plan to investigate the effect of TLR4 signaling on the regulation of cytosolic calcium concentration. Finally, we will try to elucidate the mechanisms underlying the apoptotic effect of TLR4 signaling on T cells in SAP. The present proposal may not only help us to make a better understanding of the pathophysiological mechanism of SAP, but also provide potential therapy targets for SAP.

TLR4参与重症急性胰腺炎（SAP）胰腺损伤及全身炎症反应等发病环节，但是否与SAP免疫抑制发生相关目前尚不清楚。我们研究发现：SAP T细胞TLR4和STIM1表达上调，胞浆游离钙水平（[Ca2+]i）升高且凋亡增加。因而推测TLR4可能通过影响STIM1介导钙池调控钙离子内流（SOCE），导致钙超载和T细胞凋亡参与SAP免疫抑制的发生发展。拟采用TLR4-/-、MyD88-/-和TRIF-/-小鼠，分析TLR4及下游信号分子对SAP 免疫功能和T细胞凋亡影响；阐释TLR4信号途径参与SAP免疫抑制的分子机制。本项目将深化对SAP病生理机制的认识并为SAP提供可能的治疗靶点。

课题研究分基础和临床研究两部分，已取得初步研究结果，拟在此基础上进一步开展研究工作。临床研究通过选取腹部外科32脓毒症患者及同期住院的36例非脓毒症患者作为研究对象，分脓毒症组、参芪扶正液组和对照组。入选患者记录确诊后第1、3、7天生命体征、 血常规、血生化、动脉血气分析等，进行APACHE-II评分；流式细胞仪检测外周血T淋巴细胞亚群及凋亡比例。酶联免疫吸附法(ELISA)检测血清内毒素及细胞因子水平。结果显示，与对照组相比，脓毒症组患者血炎性细胞因子IL-6、IL-8、TNF-a及细菌内毒素水平升高； CD4+T细胞比例、CD4+/CD8+比值下降，CD4+T细胞凋亡比例升高；APACHE-II评分升高；应用参芪扶正液治疗后，促炎细胞因子和细菌内毒素水平下降；CD4+T细胞比例、CD4+/CD8+比值升高，CD4+T细胞凋亡比例下降；APACHE-II评分下降。表明应用参芪扶正液治疗，通过减少CD4+T细胞的凋亡，改善CD4+/CD8+比例失衡，减轻SAP免疫紊乱和多脏器功能损害发挥治疗效应。基础研究部分，通过采用胆胰管逆行注入5％牛磺胆酸钠的方法制备大鼠SAP模型，分为SAP组、对照组和大黄素（emodin)治疗组，流式细胞仪检测Treg的数量及比例，荧光定量PCR法检测Foxp3、GITR、CTLA-4表达，ELISA法检测外周血IL-10、IL-6、IL-12、TNF-α、MCP-1及肺组织匀浆MPO水平；检测肺、肝、胰腺、肾组织病理变化，及淀粉酶、胰脂肪酶、ALT、AST、Cr、BUN等。结果显示与sham组比较，SAP组Treg数量及比例都升高（P<0.05），Foxp3、GITR、CTLA-4表达都增强（P<0.05）；与SAP组比较，Emodin治疗组Treg数量及比例降低（P<0.05），Foxp3、CTLA-4表达下降（P<0.05），表明，emodin可能通过影响Treg的凋亡和功能，改善SAP导致的免疫紊乱和多脏器功能损害，发挥治疗效应。