传染性胃肠炎病毒感染猪小肠上皮细胞上调FcRn表达的分子机制研究

The secretory IgA plays a major role in the mucosal anti-infection immunity. However, recent studies have found that IgG also plays an important role against pathogenic infection in mucosal sites, while the neonatal Fc receptor (FcRn) is the only IgG transport receptor. FcRn expression can be triggered upon stimulation with pathogenic invasion on mucosal surfaces, which may significantly enhance host mucosal defense. Our preliminary data showed that the porcine transmissible gastroenteritis virus (TGEV) stimulated porcine small intestinal epithelial cells (IPEC-J2) could increase FcRn expression, which may causes some signaling pathways activated such as NF-κB signaling pathway by specific molecular pattern recognition receptors (PRRs) or the secretion of proinflammatory cytokines. However, the accurate mechanism by which FcRn upregulated expression is not clear and remains to be elucidated. In this study, we will analyze the dynamic of FcRn expression in IPEC-J2 cells after the infection of TGEV; investigate the upstream signaling pathways of upregulated expression of FcRn induced by TGEV; unearth the proteins that invovled in the process of upreglation and located their functional domains; detect the cytokines invovled in the process; fully uncover the molecular mechanism of upregulation of FcRn expression by TGEV, confirm the upregulated expression of FcRn can enhance its function for IgG transport across the polarized epithelial cells. It will be helpful to elucidate the role of FcRn in the mucosal anti-infection immunity and FcRn bridging innate and adaptive immune responses at mucosal surfaces, reveal TGEV molecular pathogenesis and immune mechanism, provide scientific and theoretical basis for prevention and controlling of TGEV infection.

分泌型IgA在黏膜抗感染中起主要作用，但近年来发现IgG在黏膜抗感染中也发挥重要作用，而FcRn是IgG的唯一转运受体。当病原经黏膜入侵时，黏膜上皮会上调FcRn表达以增强机体抗感染的能力。我们的前期研究表明传染性胃肠炎病毒（TGEV）感染猪小肠上皮细胞（IPEC-J2）能上调FcRn表达，可能通过天然免疫模式识别受体识别病毒、分泌促炎症因子等激活相关信号通路上调FcRn表达，但其调控机制不清楚。本研究拟分析TGEV感染IPEC-J2细胞后FcRn的表达动态，探讨TGEV上调FcRn表达的上游信号通路，挖掘参与调控的TGEV编码蛋白并确定其功能域，检测促炎症因子对FcRn的上调表达，证实上调FcRn的表达能增强其转运IgG的功能，全面解析TGEV上调FcRn表达的分子机制，阐明FcRn在黏膜抗感染以及在天然免疫和适应性免疫之间所起的桥梁作用，为揭示TGEV的分子致病机理和免疫机制奠定基础。

我们的前期研究发现传染性胃肠炎病毒（TGEV）感染猪小肠上皮细胞（IPEC-J2）能够上调FcRn的表达，但其调控机制不清楚。本课题研究发现TGEV感染IPEC-J2能通过激活NF-κB调控FcRn的表达，通过构建不同长度的FcRn启动子荧光素酶报告质粒，经荧光素酶活性检测发现FcRn启动子区NF-κB主要结合位点位于上游的-1381～-208之间，进一步证实这个区域具有4个p65转录因子结合位点。通过构建TGEV病毒蛋白真核表达载体筛选调控FcRn的蛋白，发现TGEV核衣壳蛋白（N）能够通过激活NF-κB上调FcRn表达，其中心区域（aa 128–252）为关键结构域。通过干扰分子试验研究发现TGEV能通过TLR3、TLR9和RIG-1上调FcRn表达。通过分析TGEV感染IPEC-J2细胞诱导产生的炎性因子，发现TNF-α、IL-1β及 TGF-β均能上调FcRn的表达，且TGF-β通过JNK MAPK信号通路调控FcRn的表达。利用IPEC-J2细胞体外胞转模型发现FcRn能够跨越黏膜屏障转运抗TGEV的特异性IgG并起到中和病毒的作用，进一步在胞转模型上发现昆虫细胞表达的重组N蛋白能上调FcRn表达并增强IgG的转运。本研究全面解析了TGEV上调FcRn表达的分子机制，阐明了FcRn在抵抗病毒入侵及黏膜免疫中发挥的重要作用。此外我们研究发现与TGEV同属于冠状病毒的猪流行性腹泻病毒（PEDV）和猪德尔塔冠状病毒（PDCoV）能下调FcRn的表达，目前正在对其调控机制展开研究。