感染旋毛虫后小鼠腹腔巨噬细胞NOD1样受体及相关细胞因子的表达动态分析  

Expression changes of NOD1 receptor and relevant cell factors in mice peritoneal macrophages after Trichinella spiralis infection

作　　者：李晓云[1] 阴玥[1] 韩彩霞[1] 胡静 孟诗 张妍[1] 陈博[1] 刘宇琦 孟小晴 宋铭忻[1] LI Xiao-yun;YIN Yue;HAN Cai-xia;HU Jing;MENG Shi;ZHANG Yan;CHEN Bo;LIU Yu-qi;MENG Xiao-qing;SONG Ming-xin(Heilongiang Key Laboratory for Zoonosis/Northeast Agricultural University,Harbin 150030,China)

机构地区：[1]东北农业大学动物源性人兽共患病黑龙江省重点实验室

出　　处：《中国兽医科学》2018年第12期1536-1541,共6页Chinese Veterinary Science

基　　金：“十三五”国家重点研发计划项目(2017YFD0501300-2);国家寄生虫种质资源共享服务平台项目(TDRC-2017-22)

摘　　要：为了解感染旋毛虫后宿主巨噬细胞NOD1受体及其信号通路中关键分子与相关细胞因子的表达动态,将一定量旋毛虫肌幼虫经口感染小鼠,在感染后第0.5、4、7、14、21、28、35天分别取小鼠腹腔巨噬细胞,应用荧光定量PCR检测细胞中NOD1、RIP2、NF-κB mRNA表达量,Western-blot测定NOD1、RIP2、NF-κB蛋白表达量,ELISA测定细胞培养上清及血清中TNF-α、IL-1β和IL-6的质量浓度。荧光定量PCR结果显示,感染旋毛虫后巨噬细胞中NOD1、RIP2、NF-κB的mRNA表达量变化趋势基本一致,均呈现＂升高-降低-升高-降低-平稳＂的态势,且分别在感染后第4和21天各具有1个mRNA表达量峰值,在第28～35天时mRNA水平降低并趋向稳定。Western-blot结果表明,NOD1、RIP2和NF-κB p-p65的蛋白质量浓度变化趋势与其mRNA表达量检测结果基本一致。ELISA结果显示,感染小鼠腹腔巨噬细胞培养上清和小鼠血清中TNF-α、IL-6的质量浓度与对照组相比变化明显（P〈0.01,P〈0.05）,变化趋势与NOD1、RIP2和NF-κB的变化相似,IL-1β的质量浓度变化程度不明显。结果表明,旋毛虫感染可以引起小鼠NOD1受体的激活,在旋毛虫生活史的特定时期上调NOD1受体通路中关键分子RIP2和下游分子NF-κB的表达,促进细胞因子TNF-α和IL-6的分泌。试验证明,宿主NOD1受体参与了旋毛虫感染引起的免疫应答,为防治旋毛虫病和理解旋毛虫对宿主的感染机制提供了新的思路。To study the dynamic changes in expression level of NOD1 receptor,key molecules in its signaling pathway and related cytokines,the mice were orally infected with larvae of Trichinella spiralis and peritoneal macrophages were collected after infection on day 0.5,4,7,14,21,28 and 35.The mRNA levels of NOD1,RIP2 and NF-κB were assayed by q PCR.The protein expressions of NOD1,RIP2 and NF-κB were examined by Western-blot. The concentrations of TNF-α,IL-1β and IL-6 in serum and supernatant of cultured macrophages were determined by ELISA.q PCR analysis indicated the total trends of dynamic change of NOD1,RIP2 and NF-κB mRNA were basically consistent,which all showed the change of ＂increase-decrease-increase-decrease-stable＂,with a peak mRNA level respectively on day 4 and 21 post infection,then the levels tended to decline and stabilize on day 28 to 35.Western-blot results showed the change trends of NOD1,RIP2 and NF-κB p-p65 protein were similar to those of q PCR analysis of mRNA level. ELISA results indicated that the concentrations of TNF-α and IL-6 in sera and culture supernatants of macrophages showed significant differences between pre-infection and post-infection（P〈0.01,P〈0.05）,but IL-1β variation difference was small.The results indicated T. spiralis infection activated NOD1 receptor pathway in mice peritoneal macrophages,upregulated the expressions of key molecule RIP2 and downstream molecule NF-κB,and promoted the secretion of TNF-α and IL-6 during specified periods in T.spiralis life cycle.The study revealed NOD1 receptor participated in the host immune responses,and provided new data for trichinelliasis control and infection mechanism understanding.

关 键 词：旋毛虫感染 腹腔巨噬细胞 NOD1受体通路 

分 类 号：S852.731[农业科学—基础兽医学]