NOD样受体蛋白3对小鼠巨噬细胞吞噬创伤弧菌能力的影响  

Role of NOD-like receptor protein-3 in regulating phagocytosis in Vibrio vulnificus-infected murine macrophages

作　　者：楼涵 黄显辉[2,3,4] 楼永良 谢旦立[2,3,4] Lou Han;Huang Xianhui;Lou Yongliang;Xie Danli(School of Basic Medical Science,Wenzhou Medical University,Wenzhou 325025,China;School of Ijdboratory Medicine,Wenzhou Medical University,Wenzhou 325025,China;Key hiboratory of Laboratory Medicine,Ministry of Education of China,Wenzhou 325025,China;Wenzhou Key Liboratory of Sanitary Microbiology,Wenzhou 325025,China)

机构地区：[1]温州医科大学基础医学院,325025 [2]温州医科大学检验医学院,325025 [3]检验医学教育部重点实验室,温州325025 [4]溫州市环境卫生微生物捡验重点实验室,325025

出　　处：《中华微生物学和免疫学杂志》2021年第2期106-110,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金(31400764);浙江省自然科学基金(LY13H190007);温州市科技局公益性科技项目(Y20150113)。

摘　　要：目的探讨NOD样受体蛋白3(NOD-like receptor protein 3,NLRP3)对巨噬细胞吞噬创伤弧菌能力的影响和调控机制。方法转录组测序分析创伤弧菌感染J774A.1细胞吞噬相关基因的表达谱;CRISPR-Cas9基因编辑技术构建NLRP3敲除(NLRP3 KO)的J774A.1细胞;流式细胞术分析未敲除J774A.1和NLRP3 KO J774A.1细胞对创伤弧菌和pHrodo RED标记的大肠埃希菌(Escherichia coli,E.coli)修饰微球的吞噬能力;荧光定量PCR检测吞噬相关基因Fgr2b的表达情况。结果J774A.1细胞感染创伤弧菌后,吞噬功能相关基因表达谱中约有18个基因表达上调(P<0.05);NLRP3 KO J774A.1细胞在NLRP3的第2个外显子中有5个碱基的缺失,发生移码突变,导致NLRP3表达缺陷;创伤弧菌或E.coli修饰微球体外作用后,NLRP3 KO J774A.1细胞较未敲除组吞噬能力均显著增强,差异有统计学意义;与未敲除组比较,创伤弧菌感染的NLRP3 KO J774A.1细胞中Fgr2b基因的表达显著升高,差异有统计学意义。结论NLRP3能负调控巨噬细胞对创伤弧菌的吞噬功能,其作用机制可能与调控吞噬相关基因Fgr2b的表达有关。Objective To investigate the role of NOD-like receptor protein 3(NLRP3)in the regulation of phagocytosis in Vibrio vulnificus(V.vulnificus)-infected macrophages.Methods Expression profiles of phagocytosis-related genes in PBS-and V.vulnificus-infected J774A.1 cells were analyzed by RNA-Seq.NLRP3-knockout(NLRP3 KO)J774A.1 cells were constructed using CRISPR-Cas9 gene-editing system.The phagocytosis of V.vulnificus and pHrodo RED-labelled Escherichia coli(E.coli)bioparticles in parental and NLRP3 KO J774A.1 cells was detected by flow cytometry.Real-time PCR was performed to measure the expression of Fgr2b gene at mRNA level in PBS-and V.vulnificus-treated parental and NLRP3 KO J774A.1 cells.Results The expression of 18 phagocytosis-related genes was upregulated in V.vulnificus-infected J774A.1 cells than in PBS-treated J774A.1 cells(P<0.05).There was a 5 bp deletion in the exon 2 of NLRP3 gene in NLRP3 KO J774A.1 cells,resulting in frameshift mutation and complete loss of NLRP3 expression.NLRP3 KO J774A.1 cells exhibited enhanced phagocytosis of V.vulnificus and pHrodo RED-labelled E.coli bioparticles than parental J774A.1 cells(P<0.05).Besides,the expression of Fgr2b gene at mRNA level was significantly increased in V.vulnificus-infected NLRP3 KO J774A.1 cells than in parental J774A.1 cells(P<0.05).Conclusions The phagocytosis of V.vulnificus in macrophages could be negatively regulated by NLRP3,which was possibly mediated through the regulation of Fgr2b gene expression.

关 键 词：NOD样受体蛋白3 巨噬细胞 吞噬 创伤弧菌 

分 类 号：R378.3[医药卫生—病原生物学]