TNF-α诱导蛋白-8样分子2介导烧伤后免疫功能障碍及其机制研究

With both animal experiments and clinical observations, the present study was performed to investigate the potential role, regulatory mechanism and interventional strategy of tumor necrosis factor (TNF)-α induced protein 8 like-2 (TIPE2) in mediated cellular immune dysfunction after major burns, which might be of significance in understanding the novel insights into pathogenesis of abnormal host immune response in the development of postburn sepsis. The main research contents of this project include various aspects as follows: (1) to investigate the effect of TIPE2 on different types of immune cells (such as CD4+ T lymphocytes, and regulatory T cells, Tregs) and the related mechanism of receptors; (2) to study the apoptosis and its signaling pathway of abnormal immune response mediated by TIPE2 following major burns; (3) to evaluate the relationship between TIPE2 expression and the disorder of immune function as well as its clinical significance secondary to acute insults; (4) and to seek the rational methods to regulate immune response by targeting the key point and regulatory mechanism of TIPE2. From the current study, we will try to elucidate the internal relationship between TIPE2 expression and dysfunction of cell-mediated immunity, and also the biological significance as well as pathophysiological role of TIPE2 in the immunopathogenesis of severe sepsis and subsequent multiple organ dysfunction syndrome. Meanwhile, the precise receptors, signal transduction, apoptosis pathway and regulatory process of abnormal cellular immunity induced by TIPE2 could be clarified from this study, thereby proposing the new theory concerning cellular immunosuppression after severe burns. These insightful findings on TIPE2 would provide a better understanding the pathogenesis of sepsis, allow an opportunity to explore a new way to regulate inflammation and immunological responses, and develop novel immunomodulatory strategy for improving the clinical outcome of patients with extensive burns and subsequent septic complications.

该课题拟通过动物实验与临床观察，从多个层次及不同侧面探讨新的负向调控蛋白- - 肿瘤坏死因子-α诱导蛋白-8样分子2(TIPE2)在烧伤后细胞免疫功能紊乱中的可能作用、分子机制与干预措施，对于从新的视角深入认识烧伤脓毒症的发病机制具有重要理论意义和潜在临床价值。主要内容包括：探讨TIPE2对T淋巴细胞和调节性T细胞免疫应答的影响及受体机制；研究TIPE2介导免疫反应异常的凋亡途径与相关信号通路；分析TIPE2与严重烧伤后免疫功能紊乱的关系及其临床意义；围绕TIPE2寻求调节机体免疫反应的有效方法。该课题旨在明确烧伤后TIPE2表达与细胞免疫紊乱的内在联系及其在脓毒症发病中的地位；弄清TIPE2引起细胞免疫反应异常的受体与信号转导、凋亡及调节过程；提出TIPE2介导严重烧伤后细胞免疫功能障碍的新理论；通过对TIPE2进行干预的研究，探索调控机体炎症与免疫反应、预防和治疗脓毒症的新途径。

TIPE2作为一种新的负向调节蛋白参与了失控炎症反应的病理过程，但其确切免疫效应及信号机制有待探明。本课题拟探讨TIPE2在烧伤后细胞免疫功能障碍中的可能作用、分子机制与干预措施，对于从新的视角深入认识烧伤脓毒症的发病过程具有重要意义和潜在价值。主要研究结果如下：(1)激光共聚焦显微镜观察到T细胞胞浆中高表达TIPE2，烧伤小鼠脾组织TIPE2基因/蛋白表达显著上调，沉默TIPE2基因表达后T细胞增殖指数明显升高、凋亡减少，且IFN-γ/IL-4比值升高。(2)从基因和蛋白水平检测到Treg中TIPE2呈阳性表达，主要位于胞浆中；siRNA-TIPE2处理活化Treg后CTLA-4和Foxp3表达下降，IL-10和TGF-β生成量显著减少，Treg对效应T细胞的抑制功能明显减弱。(3)体内外实验证实，脾组织T细胞凋亡率在TIPE2-RNA组最高，其p-smad2/p-Smad3及凋亡蛋白Bim表达显著增强，而Bcl-2表达明显降低；TIPE2-RNA组T细胞线粒体膜电位显著下降、caspase-3/9活性增加，但在TIPE2-siRNA组其改变与之相反。(4)siRNA-TIPE2转染DC组共培养上清中检测到较高水平IL-12、TNF-α，而TIPE2-RNA转染组其水平下降。沉默或过表达DC中TIPE2可显著影响T细胞的增殖活性及分化过程。(5)严重烧伤患者Treg表面分子CTLA-4表达及IL-10分泌水平明显增多，TIPE2表达亦显著上调；烧伤脓毒症患者TIPE2平均表达水平与并发MODS、病死率增加呈显著正相关，但与外周血Treg细胞 TLR4表达强度相关性不明显。结果表明，TIPE2过度表达主要通过线粒体途径诱发T细胞凋亡，并显著影响Treg的免疫抑制效应，是介导严重烧伤后细胞免疫功能紊乱的重要调控分子之一。