CD4+T细胞向胸膜腔迁移募集参与结核性胸腔积液发生机制的研究

Tuberculous pleural effusion (TPE) is the most important sign with tuberculous pleurisy patients.TPE results from Mycobacterium tuberculosis infection of the pleura and is characterized by anintense chronic accumulation of inflammatory cells in pleural space, and CD4+ T cells are adominant population in TPE. Pleural space has visceral layer and parietal layer. Both viscerallayer and parietal layer are composed of pleural mesothelial cells (PMC). So, the function and changes of PMC should be a key point in the pleural diseases. But the role of PMC in CD4+ T cells migration cross pleura and recruitment in the pleural space is poorly understood. We have completed previously some projects about TPE. We reported for the first time that PMC are able to function as antigen-presenting cells to stimulate CD4+ T cells differentiation and recruitment. However, we do not fully understand what and how PMC change in the structure and function. Chemokines are necessary for CD4+ T cells migration across pleura. Besides the chemokines, intracellular mobility of CD4+ T cells should be same important in the process of migration. But for our knowledge there is no report about intracellular mobility of CD4+ T cells which migrate across pleura. On the other hand, studies revealed calpain is involved in cell migration. 1. Calpain mediated cleavage of intercellular junctional proteins such as occludin and E-cadherin which facilitated migration of polymorphonuclear cells across the epithelial barrier. 2. Calpain which facilitated migration of polymorphonuclear cells across the epithelial barrier. 3. Calpain can cleave adhesion complex proteins such as focal adhesion kinase and talin which result in adhesion complex turnover or detachment of the cell rear. This is a key step of cells migration. 4. Calpain can modulate intracellular cytoskeletal proteins such as actin which mediated cells spreading and membrane protrusion in the migration. Thus, calpain plays a pivotal role in many aspects of cells migration. Moreover, it was reported that calpain mediated many kinds of cells proliferation. However, the role of calpain in CD4+ T cells migration across the pleura and differentiation and proliferation in the pleural space is unknown. We did some preliminary experiments and found that calpain activity was increased in tuberculous pleurisy which should be involved in changes of permeability of the pleura, and TPE can induce changes in calpain activity in CD4+ T cells which should be involved in their migration. Based on the current research trend and our preliminary experiments, we hypothesized that calpain plays a pivotal role in CD4+ T cells migration and recruitment into pleural space in tuberculous pleurisy. So, we plan to do investigations by using cell models and clinical samples to understand the detailed mechanism of CD4+ T cells migration and recruitment into pleural space of patients with tuberculous pleurisy.

结核性胸腔积液发生时CD4+T细胞向胸膜腔内积聚。课题组前期系列研究表明：结核性胸腔积液时，胸膜间皮细胞参与CD4+T细胞向胸膜的趋化及粘附；同时CD4+T细胞及其亚群也在胸膜腔中募集。然而，对于这些细胞向胸膜腔内募集尤其细胞迁移进入胸膜的机制少有研究。另一方面，前期研究发现，钙蛋白酶参与调控细胞迁移和增殖的多个环节。我们的预实验提示“结核性胸腔积液中胸膜间皮细胞内钙蛋白酶表达显著增加；钙蛋白酶活化可导致胸膜间皮细胞表达细胞间连接蛋白（E-钙粘蛋白等）显著降低，可能直接参与胸膜通透性的提高；CD4+T细胞在结核性胸腔积液作用下细胞内钙蛋白酶活化”，因此我们提出“钙蛋白酶活化参与调控CD4+T细胞向胸膜腔内迁移并募集”的假说。本课题拟利用细胞模型和结核性胸膜炎临床标本，从CD4+T细胞的趋化、粘附、迁移、分化及增殖等环节研究CD4+T细胞向胸膜腔迁移募集的机制，可为疾病的防治提供新的靶点。

胸腔积液免疫相关的发病与防治机制是亟待解决的重要问题，从T淋巴细胞聚集的角度思考和研究可望有所突破。课题组深入探讨了NO影响胸膜血管通透性的分子生物学机制，我们利用不同浓度的IL-27刺激胸膜间皮细胞，发现IL-27可以抑制胸膜间皮细胞分泌NO。NO可促进Th1细胞的分化，抑制Th17细胞的分化，从而调节MPE中Th1/Th17细胞免疫反应，影响MPE小鼠胸膜血管通透性而抑制MPE的形成。同时，课题组探讨了大量放液前后胸腔积液患者心肺功能的变化，并发现在大量放液后即刻以及24小时后，患者的左心收缩和舒张功能得到显著改善。另外，我们回顾性分析了恶性胸腔积液患者免疫相关临床指标对预测预后的价值。结果显示血清碱性磷酸酶与胸水乳酸脱氢酶联合（AL）评分、中性粒细胞、单核细胞和血小板计数是MPE患者总生存期预后的独立影响因子。