旋毛虫ES抗原对小鼠腹腔巨噬细胞NOD1受体通路影响的研究

Impact of the Trichinella spiralis ES antigen on mouse peritoneal macrophage NOD1 receptor pathway

为了解旋毛虫ES抗原对宿主巨噬细胞NOD1受体通路的影响,本研究通过体外实验将旋毛虫ES抗原作用于小鼠腹腔巨噬细胞,观察NOD1受体及其信号通路中关键分子及相关细胞因子的表达动态。应用荧光定量PCR监测细胞内NOD1、RIP2和NF-κB m RNA的转录水平,western blot测定NOD1、RIP2、NF-κBp65、NF-κB p-p65蛋白表达量,ELISA测定细胞培养上清中TNF-α、IL-1β和IL-6含量变化。结果显示,在一定的ES抗原作用浓度和时间范围内,巨噬细胞中各目的基因的转录水平均先增高,当作用时间和作用浓度超过一定值时则开始下降,作用24 h时NOD1 m RNA转录水平显著低于对照组(p<0.01);ES抗原浓度15μg/mL作用时间9 h时,巨噬细胞中NOD1、RIP2和NF-κB p-p65蛋白量显著增加(p<0.01),此时巨噬细胞培养上清中TNF-α、IL-1β、IL-6含量显著增高(p<0.01)。结果表明,旋毛虫ES抗原在一定的作用时间和浓度范围内,可以激活并调节小鼠腹腔巨噬细胞中NOD1受体通路,上调NOD1受体通路中关键分子RIP2和下游分子NF-κB的表达,可以促进细胞因子TNF-α、IL-1β和IL-6的分泌;并且超过一定时间和浓度的ES抗原刺激可以导致小鼠腹腔巨噬细胞出现免疫耐受。本研究证明了宿主NOD1受体参与了旋毛虫引起的宿主免疫应答,为旋毛虫病防治和理解旋毛虫对宿主的感染机制提供新的思路。

To investigate the effect of excretory-secretory(ES)antigen from Trichinella spiralis on macrophages NOD1receptor pathway in vitro,the mR NA levels of NOD1,RIP2 and NF-κB were assayed by qPCR,the protein expressions of NOD1,RIP2,NF-κBp65 and NF-κB p-p65 were examined by western blot and the concentrations of TNF-α,IL-1βand IL-6 in supernatant of cultured macrophages were determined with ELISA.Results showed transcriptional levels of all target genes increased firstly,then decreased over a critical concentration or a period of time,and NOD1 mR NA level was significantly lower than those in control groups(p<0.01)after 24 hrs of interaction.NOD1,RIP2 and NF-κB p-p65 proteins significantly increased compared with control groups when ES antigen concentration were 15μg/mL and interaction time reached 9 hrs,in the mean time,cytokine levels of TNF-α,IL-1β,IL-6 in culture supernatant were higher than those in the controls(p<0.01).The results indicated T.spiralis ES antigen activated and adjusted NOD1 receptor pathway in mice macrophages in a certain range of concentrations and time,upregulated expressions of key molecule RIP2 and downstream molecule NF-κB in NOD1 receptor pathway and promoted secretion of TNF-α,IL-1βand IL-6.Additionally,our studies also showed over a critical concentration or a period of time,T.spiralis ES antigen induced immune tolerance of NOD1 receptor in host.The study revealed NOD1 receptors participates in the host immune responses,and provided new data for parasitic diseases control and infection mechanism understanding.