HEK293T细胞中NLRP3炎性小体活化体系的构建  

Reconstruction of NLRP3 Inflammasome System in HEK293T Cells

作　　者：罗玲[1] 赵锴 LUO Ling;ZHAO Kai(Department of Hematology,the Third Xiangya Hospital,Central South University,Changsha 410013,China)

机构地区：[1]中南大学湘雅三医院血液科,长沙410013

出　　处：《激光生物学报》2022年第4期331-336,共6页Acta Laser Biology Sinica

基　　金：国家重点研发计划项目(2021YFC2500802);湖南省优秀青年基金项目(2021JJ20090)。

摘　　要：NOD样受体热蛋白结构域相关蛋白3(NLRP3)炎性小体是一种蛋白复合体,在机体抵抗感染以及无菌性炎症中发挥了至关重要的作用。在HEK293T细胞中构建NLRP3炎性小体活化体系有助于研究其活化及调节机制。将质粒NLRP3、凋亡相关点样蛋白(ASC)、胱冬肽酶-1(Caspase-1)以及pro-1β转染至HEK293T细胞中,随后加入尼日利亚菌素(Nigericin),并利用Western blot以及酶联免疫吸附试验(ELISA)测定成熟的IL-1β。试验结果表明:转染了完整NLRP3炎性小体组分的细胞蛋白和上清中均可检测到IL-1β的成熟体,反之,未转染完整组分的细胞蛋白及上清中不能检测到IL-1β成熟体;同时,随着NLRP3转染量的增加,可检测到的IL-1β的成熟体增加。本研究在HEK293T细胞中成功构建了NLRP3炎性小体活化体系,为后续研究奠定了基础。NOD-like receptor thermal protein domain associated protein 3(NLRP3)inflammasome is a multiprotein complex that plays a crucial role in host immune responses to both pathogen infections and sterile injuries.Construction of the NLRP3 inflammasome system in HEK293T cells provides a more convenient and concise method for studying the regulation mechanisms of inflammasome activation.Plasmids NLRP3,apoptosis-associated speck-like protein(ASC),Caspase-1,and pro-1βwere transfected into HEK293T cells.Subsequently,the mature IL-1βlevels were determined by Western blot and enzyme-linked immunosorbent assay(ELISA)after adding nigericin.Our results showed that the mature IL-1βcould be detected in cell lysates and supernatants of cells transfected with intact NLRP3 inflammasome components whereas not in cells transfected with incomplete NLRP3 inflammasome components.Meanwhile,the mature IL-1βincreased with the elevated amounts of transfected NLRP3.In this study,NLRP3 inflammasome system was successfully constructed in HEK293T cells,which laid a foundation for future research.

关 键 词：NLRP3炎性小体 HEK293T细胞 质粒转染 IL-1β成熟体 抗感染 

分 类 号：R392[医药卫生—免疫学]