Th1/Th2型免疫反应相关基因在巨噬细胞RAW264.7应对细粒棘球绦虫原头蚴刺激时的表达谱研究

Study on the Expression Profile of Th1/Th2 Immune Response-related Genes in Macrophage RAW264.7 in Response to the Stimulation of Echinococcus granulosus Protoscoleces

本研究旨在解析巨噬细胞RAW264.7在应对细粒棘球绦虫原头蚴刺激时其Th1、Th2型免疫反应相关基因的差异表达规律,为进一步揭示巨噬细胞抗细粒棘球绦虫原头蚴免疫调控机制奠定理论基础。将细粒棘球绦虫原头蚴和巨噬细胞RAW264.7共培养6、24、72 h,收集RAW264.7细胞,提取总RNA,构建cDNA文库,利用RNA sequencing技术进行测序分析。结果表明,当细粒棘球绦虫原头蚴(PSC)与巨噬细胞RAW264.7共培养6、24、72 h后,和PBS对照组相比,PSC处理组分别有848、3745和7009个基因的表达出现显著差异变化,其中上调的基因分别为415、1159和2237个,下调的基因分别为433、2586和4772个。对Th1、Th2型免疫反应相关基因的差异表达分析结果显示,当巨噬细胞RAW264.7与细粒棘球绦虫原头蚴共培养6 h时,共有31个Th1、Th2型免疫反应相关基因的表达出现显著变化,其中15个基因出现显著上调,16个出现显著下调。当巨噬细胞RAW264.7与细粒棘球绦虫原头蚴共培养24 h时,共有111个Th1、Th2型免疫反应相关基因的表达出现显著变化,其中34个基因出现显著上调,78个出现显著下调。当巨噬细胞RAW264.7与细粒棘球绦虫原头蚴共培养72 h时,共有212个Th1、Th2型免疫反应相关基因的表达出现显著变化,其中50个基因出现显著上调,162个出现显著下调。这些差异表达的基因主要包括富亮氨酸重复序列蛋白LRRCs、G蛋白偶联受体GPCRs、C型凝集素受体CLRs、清道夫受体SRs等,同时还有一些模式识别受体PRR下游信号分子以及一些PRR下游效应分子。同时分析结果显示,当细粒棘球绦虫原头蚴与巨噬细胞RAW264.7共培养6、24 h时,其Th1型的免疫反应相关的细胞因子编码基因,诸如Tnfrsf1a、Ifnar1的Tnfrsf12a等,它们的表达显著上调;而72 h时,其Th2型免疫反应相关细胞因子编码基因,诸如IL4和IL6等,它们的表达显著上调。同时研究随机选取了部分差异表达的基因进行了qRT-PCR验证,结果表明其表达趋势与RNA-seq结果一致。本研究系统解析了巨噬细胞RAW264.7应对细粒棘球绦虫原头蚴刺激不同时间段其相关基因的差异表达谱特性,初步筛选了巨噬细胞RAW264.7应对细粒棘球绦虫原头蚴刺激固有免疫相关的基因,为进一步解析这些差异表达基因在巨噬细胞RAW264.7应对细粒棘球绦虫原头蚴刺激时发挥作用以及调控机制奠定了基础。

This study aimed to analyze the differential expression of Th1 and Th2 type immune response-related genes in macrophages RAW264.7 in response to Echinococcus granulosus protoscoleces stimulation,this study lays a theoretical foundation for further revealing the immune regulation mechanism of macrophages against protoscoleces.The protoscoleces and macrophage RAW264.7 were cultured for 6,24 and 72 hours,total RNA was extracted from RAW264.7 cells,and cDNA library was constructed.The results showed that when the protoscoleces(PSC)were cultured with macrophages RAW264.7 for 6,24,and 72 hours,compared with the PBS control group,there were 848,3745 and 7009 genes in PSC treatment group showed significant difference,in which 415,1159 and 2237 genes were up-regulated and 433,2586 and 4772 genes were down-regulated respectively.The results of differential expression analysis of Th1 and Th2 immune response-related genes showed that when macrophages RAW264.7 were co-cultured with protoscoleces for 6 hours,a total of 31 Th1 and Th2 type immune response-related genes showed significant changes in expression,including 15 genes significantly up-regulated,16 significantly down-regulated.When macrophages RAW264.7 were cultured with protoscoleces for 24 hours,there were 112 of Th1 and Th2 type immune response-related genes were significantly changed,of which 34 were up-regulated and 78 down-regulated.When macrophages RAW264.7 were cultured with protoscoleces for 72 hours,the expression of 212 Th1 and Th2 type immune response related genes changed significantly,among which 50 genes were up-regulated and 162 genes were down-regulated.These differentially expressed genes include the Leucine-rich repeat protein LRRCs,the G protein coupled receptor GPCRs,the C type Lectin receptor CLRs,the Scavenger receptor SRS,and some downstream molecules of PRR.At the same time,the results showed that the expression of Th1-related genes such as Tnfrsf1a,Ifnar1 Tnfrsf12a and so on was significantly up-regulated when the protoscoleces were cultured with macrophages RAW264.7 for 6 and 24 hours.However,when the protoscoleces were cultured with macrophages RAW264.7 for 72 hours,the expression of Th2-related genes such as IL4 and IL6 was significantly up-regulated.At the same time,some differentially expressed genes were selected at random and verified by qRT-PCR.The results showed that the expression trend was consistent with that of RNA-seq.To sum up,this study systematically analyzed the different expression profiles of genes related to macrophages RAW264.7 responding to protoscoleces stimulation in different time periods,the genes associated with macrophage RAW264.7 response to protoscoleces stimulation and innate immunity were screened,this study provides a basis for further understanding the role of these differentially expressed genes in response to protoscoleces stimulation by macrophages RAW264.7 and their regulatory mechanisms.