神经纤毛蛋白-1介导的NF-κB信号通路在脓毒症时调节性T细胞维持稳定性中的作用及其机制研究

The role of regulatory T cells (Tregs) in the cellular immunosuppression of sepsis has been paid more attention,NF-κB signaling pathway is the critical pathway to maintain the stability of Tregs.NF-κB signaling pathway has tightly contact with the regulating effect of neuropilin(Nrp)-1 mediated.Our previous study indicated that sepsis could promote the high expression of Nrp-1 on the surface of Tregs,.and its level is closely related to the prognosis of sepsis and the stability of Tregs. In view of the Nrp-1 intervention, it can reduce the stability of Tregs and improve the microenvironment of cellular immunosuppression..Based on this, we put forward the scientific hypothesis:The impact of neuropilin-1 mediated NF-κB signaling pathway on maintaining the stability of regulatory T cells and its mechanisms in sepsis.We intend to adopt the technology of RNAi and CD4+ T cells specific neuropilin-1 gene knockout mouse model,and so on, to explore the regulating effect of neuropilin-1 mediated on the stability of Tregs and NF-κB signaling pathway,the position of Nrp-1 in mediated the stability of Tregs,The position of NF-κB signaling pathway in neuropilin-1 mediated the stability of Tregs, clarifying the significance of Nrp-1 mediated regulation the stability of Tregs,and the essential relationship with NF-κB signaling pathway,in order to provide a new clue for the study of immune regulation in sepsis.

调节性T细胞(Tregs)稳定性增强是引起脓毒症细胞免疫抑制的关键,但机制不明确。申请人研究表明:脓毒症时Tregs表达神经纤毛蛋白(Nrp)-1水平与Tregs稳定性密切相关,干预Nrp-1能够降低Tregs稳定性并改善脓毒症细胞免疫抑制。据此,我们推测Nrp-1参与脓毒症Tregs维持稳定性的调节机制。课题拟体外LPS模拟感染,RNAi控制Tregs表面Nrp-1表达,给予Nrp-1激动剂或抗体干预,探讨Nrp-1对Tregs 稳定性的影响,并采用RNAi和PDTC控制Tregs内NF-κB信号通路活化水平,探讨NF-κB信号通路参与Nrp-1作用的机制,阐明Nrp-1参与调节Tregs稳定性机制;通过体内CD4+T细胞条件性Nrp-1基因敲除小鼠CLP脓毒症模型,进一步阐明Nrp-1通过NF-κB信号通路调节Tregs稳定性在脓毒症免疫调控中意义,为研究脓毒症机制与防治提供新的线索。

Nrp-1有助于维持CD4+CD25+Tregs的稳定性。模拟脓毒症，探讨了Nrp-1对CD4+CD25+Tregs稳定性和潜在的信号通路的影响。（1）体外LPS干预，并进一步Nrp-1抗体、或转染沉默Nrp-1和ikkβ、或PDTC干预脾CD4+CD25+Tregs，随后使用Nrp-1受体激动剂Sema3A干预。体内CLP建立脓毒症模型，给予Nrp-1抗体干预。检测foxp3- TSDR表达、细胞凋亡率、特征标志物、主要细胞因子，及CD4+CD25+ Tregs的NF-κB信号活性。有或没有rSema3A干预条件下，脓毒症能够增加CD4+CD25+Tregs的稳定性，主要包括增加Foxp-3 / CTLA-4 / TGF -β1表达，减少细胞凋亡和foxp3-TSDR甲基化，TGF-β1和IL-10分泌增加，增加Tregs对CD4+T细胞免疫抑制。沉默Nrp-1或抗Nrp-1可阻断rSema3a介导的LPS刺激。脓毒症提高了NF-κB的DNA结合活性，以及p-ikkβ/ikkβ和p-P65/P65的比值。沉默ikkβ或PDTC干预，可抑制CD4+CD25+Tregs的稳定性。降低Nrp-1的表达，能够通过调节NF-κB信号通路降低CD4+CD25+Tregs的稳定性，成为脓毒症免疫调节治疗的新靶点。（2）探讨Sema3A对LPS诱导的CD4+CD25+Tregs稳定性作用及机制。LPS能够增加细胞稳定性，表现为Foxp-3、CTLA-4和TGF-β1m+的mRNA及蛋白表达增加，IL-10和sTGF-β1分泌增加，细胞凋亡减少，Foxp-3-TSDR去甲基化程度增加；同时LPS可增加细胞内NF-κB信号通路的DNA结合活性，以及主要分子NF-κB 抑制蛋白激酶β（IKKβ）和p65的磷酸化水平，说明LPS增加细胞稳定性的机制与NF-κB信号通路有关。与LPS组比较，PBS并未对细胞稳定性和NF-κB信号通路产生影响；但添加rSema3A后能进一步促进细胞稳定性，并激活NF-κB信号通路。而PDTC能够抑制rSema3A增加细胞稳定性的功能，NF-κB信号通路的DNA结合活性明显抑制。LPS能通过NF-κB信号通路增加CD4+CD25+ Tregs稳定性；Sema3A能够进一步增加CD4+CD25+Tregs稳定性，并且与NF-κB信号通路有关。