PCV2感染猪血管内皮细胞在血源树突状细胞分化与致免疫耐受中作用

Porcine circovirus disease (PCVD), caused by porcine circovirus type 2 (PCV2), is the major diseases that threaten the world's pig industry. PCV2 persistent infection characteristics may lead to immune tolerance for the virus stimulation with a long time small doses, which is another reason for the immune failure. Immature dendritic cells(iDC) and plasmacytoid DCs (pDC)play an important role in the induction of immune tolerance. The adhesion between DC and endothelial cells (EC) and DC migration through the vessel wall decide DC maturation and migration. The existence of PCV2 is associated with severe degeneration of EC. PCV2 infection in vitro early can cause the reduction of the antigen-presenting capacity and immunomodulatory function of vascular endothelial cells(VEC). Clarifying the function of PCV2-infected porcine VEC on DCs differentiation and the induction of immune tolerance can improve the immune pathogenesis of PCV2 and provide a theoretical basis and new idea for the viral disease vaccine and adjuvant development..In view of this, this project will be studied around the VEC functional change and its molecules affecting DC differentiation, signaling pathway of VEC leading to DC immunogenic tolerance, proteins and cytokines related to inhibition after PCV2 infection in vitro. The research methods will include siRNA, flow cytometry, ELISA, Western blotting, confocal microscopy, chemiluminescence technology, and the mixed lymphocyte reaction. The content includes the following four parts: (1) Constructing the gene silencing VEC that influence DC differentiation(VEGF-VEC-, IL-8-VEC-) or adhesion (ICAM-VEC-, DC-SIGN-VEC-, FUT1-VEC-); The analysis of VEC phenotype, related cytokines (VEGF, GM-CSF, TGF-beta, IL-8 TNF-α and IL-6) changes, as well as the differentiation of T lymphocytes, will help illustrate the effects of PCV2 on the function of the VEC with its different gene silence after the infection of PCV2 in vitro. (2) The molecular phenotype, related cytokines (VEGFR, GM-CSF, and TGF-beta and TNF-α, IL-6)of VEC, as well as molecular changes in connection with DC-differentiation signaling pathways will be researched following co-cultured with monocyte or MoDC, aiming at exploring the differentiation mechanism of MoDC under the influence of PCV2-infected VEC differentiation-related gene silencing. (3) The phenotypic molecules, chemokines, cytokines associated regulation and secretion of pDC will be assayed, which can explain the molecular basis of pDC differentiation under the influence of PCV2-infected VEC with differentiation-related gene silencing; (4) After co-culturing with the gene silencing VEC related to adhesion, the analysis of MoDC tolerogenic molecules, adhesion and transfer molecules, signaling pathways, surface-related inhibition molecules, and its impact on Treg cells, will illustrate the molecular basis of MoDC inducing immune tolerance under the influence of virus-infected endothelial cells with adhesion-gene silencing.

猪圆环病毒2型（PCV2）持续性感染下病毒长时间少量刺激所致免疫耐受值得关注。血管内皮细胞（VEC）可影响树突状细胞（DC）分化成熟，是其致免疫耐受及疫苗免疫失败原因。本项目围绕PCV2靶细胞-VEC，在PCV2感染后对血源DC分化调控和致免疫耐受作用，利用siRNA干涉技术，构建5株影响DC分化和黏附基因沉默VEC-，即VEGF-VEC-、ICAM-VEC-、DC-SIGN-VEC-、FUT1-VEC-、IL-8-VEC-；在细胞和分子水平利用现代免疫学和分子生物学技术研究PCV2感染后VEC表型和细胞因子、与单核细胞或DC共培养后VEC表型和细胞因子、与DC分化相关信号通路分子，及DC表型、粘附与转移、信号通路分子、VEGFR等抑制分子变化和对T细胞亚群影响，解析DC在PCV2感染内皮细胞作用下分化与致免疫耐受分子基础，既完善PCV2免疫致病机理，也为病毒病疫苗和佐剂研发提供新的思路。

猪圆环病毒2型（porcine circovirus type 2, PCV2）是引起仔猪断奶后多系统衰竭综合征的主要原发性病原。血管内皮细胞（Vascular Endothelial Cell, VEC）作为一种多功能的分泌细胞，与机体内环境联系密切，同时也是PCV2重要的靶细胞。本研究建立了猪外周血单核细胞磁珠纯化体系及诱导DC的方法，摸索出单核细胞分化诱导液中刺激因子终浓度分别为GM-CSF 100ng/ml和IL-4 1ug/ml；PCV2体外感染VEC，分不同时间点收毒，利用实时荧光定量方法检测病毒在VEC中增殖情况，显示4h即可在VEC中检测到病毒核酸，且72h持续存在，但病毒未见大量复制；将VEC中IL-8基因沉默后接毒，利用实时荧光定量方法，检测细胞中各免疫调节因子变化规律，显示PCV2感染的VEC中IL-8的表达影响其他免疫调节分子表达，抑制其表达后，病毒感染的VEC中M-CSF、ICAM-1、VCAM-1 mRNA的表达部分时间段抑制，对VEGF mRNA的表达表现部分时间段上调。这些结果提示PCV2感染对VEC的炎性趋化、粘附能力以及DC的分化、成熟等免疫调节功能可能有一定影响，并且IL-8在PCV2感染VEC中起重要作用。目前正在进行PCV2感染基因沉默的VEC对单核细胞分化部分实验，部分数据在整理中。该研究成果将为进一步揭示PCV2感染机体的发病机制提供科学依据。