PCV2诱导的猪内皮源IL-8调控单核细胞源树突状细胞分化及其信号机制

Porcine circovirus disease (PCVD) is an immunosuppressive disease. The pathogen is PCV2, whose target cell is endothelial cells(VEC).This project aims to the differentiation of endothial derived IL-8 on monocyte derived dendritic cells based on the phenotype，function and the NF-κB signaling molecule(EGFR、PI3K、Akt、IKKs、IκBα、NF-κBp65) related to the differentiation of DC. Coculture of infected-PCV2 VEC with DC is completed with Transwell. The treatment methods to the endothial derived IL-8 and the key molecules of NF-κB signaling pathway include gene silencing, overexpression, and specific antibody/inhibitor. And the related cytokines expressed by DC and the DC function will also be analyzed with flow cytometry, confocal, Western-blot, ELISA, FQ RT-PCR and lymphocyte activation experiment. The changes of DC differentiation related signaling pathways and function are explored after VEC and DC co-culture. The molecular basis of effects of endothial derived IL-8 on the differentiation and function of DC will be discussed. Based on endothial derived IL-8 and DC, mechanism of immune cell function disorder will be explained in three aspects of cells, proteins and signaling molecules, which will provide a theoretical basis and a new idea for the design of vaccine adjuvant of immunosuppressive viral diseases and the screening of a new pharmacological target for treatment.

猪圆环病毒病是免疫抑制性疾病，内皮细胞是病原PCV2靶细胞。本项目拟从PCV2感染产生内皮源IL-8对单核源树突状细胞（DC）分化调控着手，围绕DC分化表型、功能以及信号通路NF-κB中关键分子（EGFR、PI3K、Akt、IKKs、IκBα、NF-κBp65），对DC采用基因沉默、过表达、信号通路抑制剂处理等，利用Transwell将经IL-8过表达、基因沉默及抗体阻断VEC感染病毒后，分别与DC共培养，应用流式细胞术、荧光共聚焦、Western-blot、ELISA、荧光定量PCR等技术，分析病毒感染下内皮源IL-8影响DC分化的相关信号通路分子及功能变化，解析内皮源IL-8对DC分化与功能影响的分子机理，从内皮源IL-8和树突状细胞角度，在细胞、蛋白和信号分子三方面阐述PCV2感染引起免疫细胞功能紊乱机理，为免疫抑制性病毒病疫苗佐剂设计、治疗用药物新靶点研发提供理论基础与新的思路。

PCV2感染内皮细胞功能变化以及其诱导产生内皮源IL-8对单核源树突状细胞功能调控分子机理值得探索。本项目利用Transwell将经IL-8过表达、基因沉默及抗体阻断VEC感染病毒后，分别与DC共培养，应用基因表达谱、流式细胞术、荧光共聚焦、Western-blot、ELISA、荧光定量PCR等技术，检测PCV2感染产生内皮源IL-8作用后单核源树突状细胞（DC）分化表型、功能以及信号通路NF-κB中关键分子（EGFR、PI3K、Akt、IKKs、IκBα、NF-κBp65）等变化。与对照组相比，PCV2感染可上调PIEC IL-的分泌，该分泌是通过RIG-I/MDA-5/MAVS/JNK/NF-κB通路调控的；PCV2感染上调的IL-8抑制MoDC免疫反应、抗原递呈、炎性反应、血管形成、细胞粘附等功能相关基因的表达，在蛋白和细胞水平显示抑制MoDC的成熟、粘附功能、迁移能力，促进MoDC的吞噬能力；PCV2诱导的内皮源IL-8进一步促进MoDC倾向Th2免疫应答，降低对T淋巴细胞增殖和刺激Treg细胞分化能力。此外，PCV2诱导的内皮源IL-8通过抑制MoDC中IκBα的激活及降解与NF-κB p65激活，导致NF-κBp65核转移受阻以及抑制MoDC 相关细胞因子IL-12、GM-CSF的表达，从而抑制MoDC的成熟。该研究成果解析内皮源 IL-8 对DC分化与功能影响的分子机理，从内皮源 IL-8 和树突状细胞角度，在细胞、蛋白和信号分子三方面阐述 PCV2 感染引起免疫细胞功能紊乱机理，为免疫抑制性病毒病疫苗佐剂设计、治疗用药物新靶点研发提供理论基础与新的思路。