Pim-1信号在炎症性肠病发病机制中的免疫调节作用研究

Inflammatory bowel disease (IBD) is characterized by chronic relapsing inflammatory disorders of the gastrointestinal tract. Although the etiology of IBD remains unclear, accumulating evidence has indicated that dysfunction of the mucosal immune system plays an important role in the pathogenesis of IBD，Pim-1 is a serine/threonine kinase that is crucially involved in the control of cell growth, differentiation and apoptosis. Accumulating data support that Pim-1 are essential components of an endogenous pathway that regulates T cell growth and survival. The crucial role of Pim kinase in immune cells activation and proliferation makes it an attractive target for immunomodulatory therapy. Indeed, the pharmacological inhibition of Pim kinases exhibits immunomodulatory benefits, However, it remains unclear whether pim-1 kinase is involved in the pathology of IBD and whether pim-1 kinase could modulate T help cell differentiation and macrophage activation during the development of IBD. .The aims of this study were to explore the role of Pim-1 kinase in the pathology of IBD and to assess whether Pim-1 kinase may act to regulate innate and adaptive immune responses,inhibiting Pim-1 kinase may be of therapeutic benefit as a treatment regimen for IBD. In vitro effects of Pim-1 on LPS-induced macrophage activation and Th cell differentiation,as well as in vivo effects of PIM-Inhibitor (PIM-Inh) in mice with TNBS or DSS-induced colitis, were explored. In acute colitis, daily PIM-Inh treatment commenced 2 days after TNBS delivery, while in relapsing colitis PIM-Inh treatment commenced after three weekly TNBS or DSS administrations. Colon inflammation, production of proinflammatory cytokines and NF-κB activation in colon tissues,and Th1/Th2,Th17/Treg cell populations in lamina propria mononuclear cells (LPMCs) and mesenteric lymph node cells (MLNs) were assayed..In summary, our data may provide evidence that Pim-1 may act as a central regulator in the immune response by regulates macrophages activation and Th cells differentiation.These findings may offer a promising alternative to our current approaches of managing IBD.

炎症性肠病（IBD）的发病与肠粘膜免疫紊乱有关，但是相关信号分子如何调节免疫细胞功能目前还不清楚。我们前期实验中发现Pim-1信号参与小鼠结肠炎炎症的启动，并且与免疫细胞功能状态有关，阻断Pim-1信号可以抑制肠道炎症反应，根据这一结果我们提出假设：Pim-1信号参与IBD肠粘膜固有免疫和适应性免疫功能的调节。本课题以研究巨噬细胞、na?veT细胞激活过程中Pim-1表达调控的信号分子为切入点，进一步研究Pim-1在巨噬细胞激活、Th1/Th2、Treg/Th17免疫平衡调节方面的作用及机制，同时利用IBD模型，探讨体内抑制Pim-1信号对肠道异常免疫反应的影响及机制。本项目从分子、细胞、动物多方面来探讨Pim-1信号对IBD肠粘膜固有及适应性免疫细胞功能的调节作用，以阐明Pim-1信号参与肠道异常免疫反应的机制，旨在为IBD的治疗提供新的靶点，为利用Pim-1信号治疗IBD打下理论基础。

免疫因素在炎症性肠病(IBD)中重要作用已被广大学者所公认，Pim-1信号在细胞增殖、分化、凋亡等过程中起重要作用，新近研究提示Pim-1信号参与免疫调节，但是目前关于Pim-1信号是否能调节IBD肠粘膜固有及适应性免疫细胞功能的研究国内外尚未见报道，本课题成功分离巨噬细胞及naïve CD4+T细胞，并分别观察激活后Pim-1的表达，1h后即可检测Pim-1mRNA、Pim-1蛋白表达增高，高峰出现在2-24h，分别阻断PI3K、P38MAPK、JAK2、MEK1/2信号后均引起活化的巨噬细胞及naïve CD4+T细胞中Pim-1蛋白表达下调，提示Pim-1可能是PI3K、p38MAPK、JAK2、MEK1/2的下游信号分子。其中P38抑制剂、MEK1/2抑制剂抑制Pim-1表达作用更加明显。在LPS刺激前给予Pim-1抑制剂(PIM-Inh)，可以明显降低巨噬细胞NF-κBp65、iNOS，TNFα蛋白的表达，且呈剂量依赖性；本课题成功诱导分化免疫磁珠（MACS）分离的naïve CD4+T细胞，使用Pim-1抑制剂可以抑制naive CD4+T细胞T-bet、RORγt的表达，并降低上清液中IFN-γ、L-17的水平，采用流式细胞检测细胞亚群，Pim-1抑制剂可以抑制naive CD4+T细胞向Th1、Th17分化。本课题成功建立DSS,TNBS建立急性及慢性动物模型；PIM-Inh治疗组小鼠血便、腹泻症状、DAI评分、病理组织学评分较模型组均好转，PIM-Inh治疗组肠组织中T-bet、ROR-γt、IFNγ、IL17表达下降，其中高剂量治疗组下降更加明显；治疗组肠组织中p-NF-κB P65， iNOS的表达较模型组明显下降，分离正常对照组、治疗组与模型组LPMC，MLN淋巴细胞，采用胞内细胞因子荧光染色法测定Th亚群，治疗组Th1、Th17细胞比率较模型组下降.结果： Pim-1信号参与巨细胞及naïveT细胞激活及分化， PI3K、p38MAPK、JAK2、MEK1/2信号可以调控的Pim-1的表达。抑制Pim-1信号可以减轻肠道炎症，机制可能与抑制CD4+T细胞向Th1、Th17分化、抑制巨噬细胞TLR4/NF-κB 信号，本课题从动物、细胞、分子水平多方面来证实Pim-1信号对IBD肠粘膜固有及适应性免疫细胞具有调节作用。