LSEC、Raw264.7、Hepa1-6三种鼠源细胞系cGAS-STING信号通路的本底表达及激活研究  

Background expression and activation of cGAS-STING signaling pathways in LSEC,Raw264.7 and Hepa1-6 cells murine cell lines

作　　者：周钰婷 张勇 杨建[1] 李飞 ZHOU Yuting;ZHANG Yong;YANG Jian;LI fei(College of Pharmacy,Xinjiang Medical University,Urumqi 830011,China;Guangxi Key Laboratory of Molecular Medicine of Liver Injury and Repair,Affiliated Hospital of Guilin Medical University;Guangxi Key Laboratory of Basic Research on Diseases Related to Nerve Sphingolipids Metabolism,Affiliated Hospital of Guilin Medical College,Guilin Guangxi 541001,China)

机构地区：[1]新疆医科大学药学院,乌鲁木齐830011 [2]桂林医学院附属医院广西肝脏损伤与修复分子医学重点实验室 [3]桂林医学院附属医院广西神经鞘脂代谢相关疾病基础研究重点实验室,广西桂林541001

出　　处：《新疆医科大学学报》2022年第5期486-491,共6页Journal of Xinjiang Medical University

基　　金：国家自然科学基金面上项目(81873185)。

摘　　要：目的研究小鼠肝窦内皮细胞(LSEC)、小鼠巨噬细胞(Raw264.7)和小鼠肝癌细胞(Hepa1-6)cGAS-STING信号通路的本底表达及激活感染情况。方法将LSEC、Raw264.7和Hepa1-6三种细胞培养过夜,使用激活剂poly(dA:dT)、2,5-己酮可可碱(DMXAA)刺激,人肝癌细胞(HepG2.2.15)上清液浓缩纯化后的乙型肝炎病毒(HBV)感染,12 h后收集细胞提取RNA和蛋白,采用实时荧光定量PCR(Q-PCR)和蛋白质免疫印迹法(Western blot)检测LSEC、Raw264.7、Hepa1-6细胞cGAS-STING信号通路关键基因cGAS、STING、IFN-β的mRNA水平和cGAS、STING、IRF3及磷酸化IRF3蛋白水平。结果LSEC的本底表达量最高。Poly(dA:dT)可显著提高3种细胞IFN-βmRNA的表达(P<0.01);DMXAA可显著提高三种细胞cGAS、STING mRNA的表达(P<0.05),促进了STING和IRF3蛋白的磷酸化。HBV可显著抑制Raw264.7、Hepa1-6细胞IFN-βmRNA的表达(P<0.01),显著激活LSEC细胞的cGAS、IFN-βmRNA的表达(P<0.01)并促进STING和IRF3蛋白的磷酸化。结论三种细胞cGAS-STING信号通路的本底表达、激活、感染存在明显差异。Objective To study the Background and activation expression of cGAS-STING signaling pathways in LSEC,Raw264.7 and Hepa1-6 cells murine cell lines with infection conditions.Methods The above three cells cultured overnight and treated with activator poly(dA:dT),2,5-hexanone cocoa base(DMXAA)and infected with hepatitis B virus(HBV)concentrated from HepG2.2.15 cell supernatant.After 12 h,the cells were collected to extract RNA and protein,respectively,and the RNA levels of cGAS,STING,IFN-β,key genes of cGAS-STING signaling pathway and cGAS,STING,IRF3 and phosphorylated IRF3 protein were detected by Q-PCR and Western blot in LSEC,Raw264.7 and Hepa1-6 cells.Results It was significantly increased on expression of IFN-βmRNA in the three cells(P<0.01);DMXAA was significantly increased on expression of cGAS,STING mRNA in the three cells(P<0.05)and promoted the phosphorylation of STING and IRF3 proteins.HBV significantly inhibited the expression of IFN-βmRNA expression(P<0.01),significantly activated the expression of cGAS,IFN-βmRNA(P<0.01)and promoted the phosphorylation of STING and IRF3 proteins in LSEC.Conclusion There were significant differences in basal expression,activation,and infection of cGAS-STING signaling pathway of three cells.

关 键 词：cGAS-STING信号通路 STING激活剂 肝细胞系 

分 类 号：R392[医药卫生—免疫学]