CD103介导的Th9细胞归巢在脓毒症肠粘膜屏障损伤中的作用研究

The intestinal mucosal barrier injury is an important etiological factor of immune imbalance in sepsis. Our previous studies found that the proportions of T helper cells were imbalanced in the early stage of sepsis, and enteral nutrition could improve this imbalance. These results indicated that intestinal T helper cells played important roles in the mechanisms of intestinal mucosal barrier injury. Th9 cell is a latest discovered T helper cell subset that played its role mainly through IL-9 pathway, it is significantly correlated with intestinal local inflammation. Our preliminary data showed that the Th9 and IL-9 levels of septic patients were significantly increased. Recent studies revealed that integrin CD103 (αEβ7) was an important vector mediated the Th9 cells homing, and overexpression of αEβ7 was closely related to intestinal inflammation. However, whether the intestinal mucosal barrier injury of sepsis is associated with this disordered αEβ7-mediated Th9/IL-9 pathway, and whether enteral nutrition could protect the mucosal barrier through this pathway are unclear. Therefore, we aim to evaluate the roles of CD103-mediated Th9 cells homing in the intestinal mucosal barrier injury and enteral nutrition treatment of sepsis, using MACS, 16s rDNA sequencing, and other technologies in vitro and in vivo studies. The results of this study would clarify a new mechanism of intestinal mucosal barrier injury, as well as find a new target for early intervention of barrier injury and immune imbalance in sepsis.

肠粘膜屏障损伤是导致脓毒症免疫失衡的重要因素，课题组前期研究发现脓毒症存在Th细胞比例失衡，而肠内营养可改善这种失衡，提示肠粘膜屏障损伤与肠道Th细胞有关。Th9细胞是新近发现的一种重要Th细胞，主要通过IL-9发挥效应，在肠道局部炎症过程中发挥关键作用。我们的预实验也发现脓毒症患者存在Th9、IL-9水平的显著升高。最新研究证实CD103（αEβ7）是介导Th9细胞归巢的重要载体，αEβ7过表达与肠道炎症密切相关，但αEβ7介导的Th9/IL-9失衡是否导致脓毒症肠粘膜屏障损伤尚不清楚，而肠内营养能否通过αEβ7、Th9改善肠粘膜屏障损伤也尚待证实。本课题旨在应用细胞及动物模型，采用免疫磁珠分选、16s rDNA测序等技术，探讨CD103介导的Th9细胞归巢在脓毒症肠粘膜屏障损伤及肠内营养治疗中的作用，明确脓毒症肠粘膜屏障损伤的新机制，为早期干预肠粘膜屏障损伤及脓毒症免疫失衡寻找新靶点。

肠粘膜屏障损伤是导致脓毒症免疫失衡的重要因素，前期研究发现肠粘膜屏障损伤与肠道Th细胞有关。Th9细胞是新近发现的一种重要Th细胞，主要通过IL-9发挥效应，在肠道局部炎症过程中发挥关键作用。最新研究证实CD103（αEβ7）是介导Th9细胞归巢的重要载体，αEβ7过表达与肠道炎症密切相关，但αEβ7介导的Th9/IL-9失衡是否导致脓毒症肠粘膜屏障损伤尚不清楚。本课题应用细胞及动物模型，采用免疫磁珠分选、Western Blot、16s rDNA测序、腺相关病毒等技术，探讨CD103（αEβ7）介导的Th9细胞归巢在脓毒症肠粘膜屏障损伤中的作用。其中，细胞实验发现，给予脂多糖（LPS）刺激后αEβ7、E-钙粘蛋白和IL-9水平上升，ZO-1水平显著下降，Caco-2细胞凋亡增加；给予IL-9蛋白或抗体后，四个指标的表达水平及细胞凋亡情况均出现相反趋势。Caco-2细胞与Th9细胞共培养，给予蛋白抗体后，αEβ7、E-钙粘蛋白和IL-9水平较单纯LPS组明显下降，ZO-1水平显著上升，细胞凋亡减少；而给予重组蛋白后，αEβ7、E-钙粘蛋白和IL-9水平较单纯LPS组明显上升，ZO-1水平显著下降，细胞凋亡增加。动物实验发现，脓毒症组大鼠的Th9 细胞比，αEβ7、E-钙粘蛋白和IL-9水平均较对照组显著上升，ZO-1水平下降；肠道组织损伤加重，通透性增加。而给予干扰腺相关病毒后，大鼠的Th9细胞比，αEβ7、E-钙粘蛋白和IL-9水平均较脓毒症组下降，ZO-1水平上升；肠道组织损伤减轻，通透性有所改善。给予过表达腺相关病毒后，大鼠的Th9细胞比，αEβ7、E-钙粘蛋白和IL-9水平均较脓毒症组上升，ZO-1水平下降；肠道组织损伤加重，通透性进一步增加。这些结果提示αEβ7、Th9和IL-9在脓毒症肠粘膜屏障损伤中发挥促进作用，阐明了脓毒症肠粘膜屏障损伤的新机制，为治疗肠粘膜屏障损伤提供了新靶点。