基于转录组测序分析细粒棘球绦虫原头蚴与巨噬细胞RAW264.7免疫互作相关基因

doi:10.11843/j.issn.0366-6964.2022.11.023

摘要/Abstract

摘要： 旨在解析细粒棘球绦虫原头蚴与巨噬细胞RAW264.7免疫互作相关的基因，为进一步阐明细粒棘球绦虫原头蚴调控宿主免疫反应及寄生适应机制提供理论依据。将细粒棘球绦虫原头蚴和巨噬细胞RAW264.7共培养24 h，收集原头蚴和RAW264.7细胞，提取总RNA，构建cDNA文库，利用RNA sequencing技术进行转录组测序分析。当细粒棘球绦虫原头蚴与巨噬细胞RAW264.7共培养24 h后，和0 h对照组相比，原头蚴处理组分别有435个基因的表达出现显著差异变化，其中，上调表达的基因为227个，包括HSP70、HSP10、Eg95前体分子、AgB、FABP和囊泡运输蛋白SC22B等；下调表达的基因为208个，包括EF-hand蛋白、Cathepsin、跨膜蛋白144、内固醇类受体和MAPK7等。GO分析结果表明，差异表达的基因主要富集在血红素转运、铁配位实体运输、细胞外间质、核糖核酸酶MRP复合物、丝氨酸型内肽酶抑制剂活性以及α-半乳糖苷酶活性等过程。KEGG分析结果表明，差异表达的基因主要参与剪接体、内吞作用、内质网的蛋白质处理、吞噬体、MAPK信号通路以及钙信号通路等。当巨噬细胞RAW264.7与细粒棘球绦虫原头蚴共培养24 h后，和PBS对照组相比，原头蚴处理组的RAW264.7细胞共有3 745个基因的表达出现显著变化，其中，1 159个基因出现上调表达，2 586个基因出现下调表达。GO分析结果显示，差异表达基因主要富集在代谢过程、细胞内组分、细胞内、细胞器以及膜结构细胞器等。KEGG信号通路的分析结果表明，差异表达的基因主要参与代谢通路、核糖体通路、剪接体通路、RNA转运以及泛素介导的蛋白水解等通路。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。综上所述，当细粒棘球绦虫原头蚴面对宿主巨噬细胞免疫压力时，可引起其基因的差异表达，其中，原头蚴中的AgB、FABP1和Kunitz型丝氨酸蛋白酶抑制剂等具有免疫调节作用的分子表达明显上调，推测其可能参与宿主的免疫调控，进而有利于虫体在宿主的寄生和免疫逃避。

关键词: 细粒棘球绦虫, 巨噬细胞RAW264.7, 差异表达, 免疫调控

Abstract: In this study, we report the analysis of the genes involved in the immune interaction between Echinococcus granulosus protoscoleces and macrophage RAW264.7, which provides a theoretical basis for further elucidating the mechanism of immune regulation and parasitic adaptation of the protoscoleces. The protoscolices and macrophage RAW264.7 cells were co-cultured for 24 hours, then the protoscolices were collected, total RNA was extracted and cDNA library was constructed. The results showed that there were 435 genes significantly differentially expressed in the protoscolices after co-cultured with macrophages RAW264.7 for 24 h compared with 0 h, which included 227 up-regulated genes, such as HSP70, HSP10, EG95 precursor, AgB, FABP and vesicular transporter SC22B; And 208 down-regulated genes, such as EF-hand, Cathepsin, transmembrane protein 144, endosterol receptor and MAPK7. GO analysis showed that the differentially expressed genes (DEGs) were mainly involved in heme transport, iron coordinated entity transport, extracellular Matrix, Ribonuclease MRP complex, serine type endopeptidase inhibitor activity and Galactosidases activity. KEGG analysis showed that the DEGs were mainly related to spliceosome, endocytosis, endoplasmic reticulum protein processing, phagosomes, MAPK signaling and calcium signaling. When macrophages RAW264.7 were co-cultured with protoscolex for 24 hours, compared with PBS control group, the expression of 3 745 genes in RAW264.7 cells was significantly changed, of which 1 159 genes were up-regulated, 2 586 genes down-regulated. The results of GO analysis showed that the DEGs were mainly concentrated in metabolic processes, intracellular components, intracellular, Organelles and membrane-structured Organelles. The analysis of KEGG showed that the DEGs were mainly involved in metabolic pathway, ribosome pathway, spliceosome pathway, RNA transport and ubiquitin-mediated protein hydrolysis. At the same time, some DEGs were selected randomly and verified by qRT-PCR. The results showed that the expression trend was consistent with that of RNA-seq results, which further indicates that the transcriptome data is credible. In conclusion, when the protoscoleces are subjected to immunologic pressure from host macrophages, they may cause gene differential expression, the immunoregulatory molecules such as AgB, FABP1 and Kunitz-type Serine protease inhibitors in protoscoleces were up-regulated, which might be involved in host immunoregulation, moreover, it is beneficial to parasitism and immune escape for E.granulosus.

Key words: Echinococcus granulosus, macrophages RAW264.7, differential expression, immune regulation

中图分类号:

S852.734

王正荣, 马勋, 张艳艳, 孟季蒙, 薄新文. 基于转录组测序分析细粒棘球绦虫原头蚴与巨噬细胞RAW264.7免疫互作相关基因[J]. 畜牧兽医学报, 2022, 53(11): 3975-3988.

WANG Zhengrong, MA Xun, ZHANG Yanyan, MENG Jimeng, BO Xinwen. Analysis of Genes Related to Immune Interaction between Protoscolices of Echinococcus granulosus and Macrophage RAW264.7 by Transcriptome Sequencing[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(11): 3975-3988.

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