生物钟基因Per2对炎症性肠病肠黏膜CD4+T细胞的调控作用及机制研究

Circadian rhythm disorders can lead to dysfunction of the intestinal mucosal immune system and cause destruction of the mucosal barrier, which suggests that the circadian clock is involved in the development of IBD. However, the mechanism still remains unclear. Per2 is one of the important circadian genes that play key roles in regulating cell proliferation, apoptosis and intestinal mucosal epithelial homeostasis. Currently, the question as to whether Per2 is involved in immune-mediated inflammatory disorders of the intestine in patients with IBD still remains to be elucidated. In our preliminary experiments, we found that Per2 expression was significantly lower in intestinal and peripheral blood mononuclear cells of patients with ulcerative colitis (UC). Further studies showed that Per2 expression also significantly decreased in UC peripheral blood CD4+T cells. In order to investigate this phenomenon, we conducted further experiments and found that the interference of Per2 with Per2-shRNA could significantly promote the expression of T-bet and IFN-γ in CD4+T cells. These findings suggested that Per2 may be involved in CD4+T cell-mediated immunopathological damage in the intestine of UC patients. To verify our hypothesis, we aim to construct CD4+T cell Per2-conditional knockout mice in order to determine how Per2 affects the proliferation and differentiation of CD4+T cells. We additionally investigate the immunomodulatory effect of Per2 in experimental mice colitis models and also study the specific mechanism of how Per2 regulates the differentiation and function of CD4+T cells. This work will shed light on the immunomodulatory effects of Per2 in colitis and provide new therapeutic targets for IBD.

生物钟节律紊乱可导致肠道免疫功能失调，引起黏膜屏障破坏，参与炎症性肠病（IBD）发生，但具体机制尚不清楚。Per2是一种重要的生物钟基因，研究报道其在调控细胞增殖、凋亡及肠黏膜上皮稳态方面发挥重要作用，但Per2是否参与IBD肠黏膜炎症损伤尚不明确。我们前期研究发现Per2在IBD特别是溃疡性结肠炎（UC）患者肠黏膜组织以及外周血单个核细胞中表达显著降低。进一步研究表明，Per2在UC患者外周血CD4+T细胞中显著下调，采用shRNA干扰Per2可明显促进CD4+T细胞中T-bet和IFN-γ的表达。上述结果提示Per2参与IBD肠黏膜组织CD4+T细胞介导的免疫损伤。为验证此假说，我们拟构建CD4+T细胞Per2特异性敲除小鼠，探究Per2对CD4+T细胞增殖与分化的影响及具体机制以及Per2在小鼠结肠炎中的免疫调节作用，阐明Per2在肠道中的免疫调节机制，为IBD治疗提供新思路。

研究背景:.生物钟节律紊乱可导致肠道免疫功能失调，引起黏膜屏障破坏，参与炎症性肠病（Inflammatory bowel disease, IBD）包括溃疡性结肠炎（Ulcerative colitis, UC）和克罗恩病（Crohn’s disease, CD）的发生，但具体机制尚不清楚。Per2是一种重要的生物钟基因，研究报道其在调控细胞增殖、凋亡及肠黏膜上皮稳态方面发挥重要作用，但Per2是否参与IBD肠黏膜炎症损伤尚不明确。.主要研究内容:.探讨Per2在IBD特别是UC发病过程中的免疫机制，我们重点研究Per2在UC患者肠黏膜组织内的表达水平、细胞亚型分布、对肠黏膜组织内CD4+T细胞的增殖分化的调节以及对其下游靶基因的调控。.重要结果以及关键数据:.1.PER2表达在活动期IBD患者特别是活动期UC患者外周血单个核细胞，肠粘膜组织以及外周血CD4+T细胞中表达降低。.通过收集IBD患者外周血单个核细胞（Peripheral blood mononuclear cell，PBMC），肠粘膜组织以及提取外周血CD4+T细胞，通过qRTPCR检测，证实PER2基因在活动期UC病人以及活动期CD病人PBMC以及外周血CD4+T细胞以及肠粘膜组织中均降低（P＜0.05），并其中以UC患者降低最明显。.2.PER2在活动期UC患者CD4+T细胞蛋白水平表达下调。.通过分离结肠癌癌旁组织以及健康者外周血不同细胞亚型发现，CD4+T在细胞各细胞亚型中表达水平较高。通过免疫组化染色，我们的研究发现，PER2在活动期UC病人中表达降低。通过免疫荧光染色以及流式细胞进一步证实，PER2在活动期UC病人中表达显著降低。.3. PER2在CD4+T细胞过表达后降低ADAM12的水平.通过慢病毒转染,使PER2在CD4+T细胞中过表达，经mRNA测序后发现，PER2过表达后过表达后降低ADAM12的表达水平，而ADAM12在Th1细胞的分化中起到重要的调控作用。.4.PER2能够调控ADAM12的表达。.通过CUT&TAG检测提示PER2能够结合到ADAM12基因内含子，进而调控ADAM12的表达。.科学意义：.通过上述研究，我们认为PER2可能是IBD治疗的新靶点，可以为临床IBD患者的治疗提供新的思路。