LPLUNC1对上呼吸道抗感染防御机制的影响

目的:拟以LPLUNC1作为上呼吸道抗感染的候选分子,探讨其在宿主细胞内抗感染防御的作用机制.方法:应用GST蛋白纯化法获取LPLUNC1融合蛋白,并采用微量肉汤稀释法测定LPLUNC1蛋白对革兰阴性菌绿脓杆菌细菌集落形成的影响;通过体外脂多糖(lipopolysaccharides,LPS)结合实验、细胞凋亡测定法探讨LPLUNC1体外结合绿脓杆菌LPS成分的能力和诱导靶细胞发生细胞凋亡的程度,并通过ELISA、Western blot印记实验分析了解LPLUNC1-LPS的结合对小鼠RAW 264.7靶细胞膜CD14受体以及胞内细胞因子TNF-α表达的影响.结果:LPLUNC1没有明显的直接抑制革兰阴性菌的特性,也不具备明显的直接杀伤革兰阴性菌的功能,但它可与革兰阴性菌LPS成分特异结合,对LPS有高度敏感性.LPLUNC1与LPS结合后可抑制CD14受体和TNF-α的表达.结论:LPLUNC1通过高度结合革兰阴性菌LPS成分,阻抑CD14介导的信号转导途径,抑制细胞内TNF-α等细胞因子的表达,引发宿主上呼吸道抗感染防御.

Effect for Signaling Mechanisms of Host Defense to Upper Respiratory Airway by LPLUNC1

Objective:To explore its signaling mechanisms of LPLUNC1 as a novel candidate member of host defense against infection in upper respiratory airway.Methods: GST pull-down method was used to amplify and purify LPLUNC1 protein.By in vitro LPS binding and cell death assay,the ability of LPLUNC1 for binding with LPS and the extent of induction to apoptosis was tested.With ELISA and Western blot assay,the expression levels of CD14 and TNF-α induction by LPLUNC1-LPS was determined.Results: In vitro LPS binding assay was verified that LPLUNC1 protein shown stronger binding ability to LPS.But in bacteria colony formation and cell death assay experiments,LPLUNC1 didn’t express direct anti-bactericidal activity and couldn’t induce cell apoptosis.When incubated with RAW 264.7 cells,LPLUNC1-LPS induction significantly inhibited the expression of CD14 and TNF-α.Conclusion:LPLUNC1 can combine with LPS,block CD14-mediated signal transduction pathway,restrain intracellular cytokines expression such as TNF-α,eventually lead to host defense against infection in upper respiratory airway.