摘要
目的:研究原代人脐静脉内皮细胞(primary human umbilical vein endothelial cells,HUVEC)被登革病毒2型(dengue virus type 2,DENV2)感染后,巨噬细胞迁移抑制因子(macrophage migration inhibitory factor,MIF)对腺苷酸激活蛋白激酶(adenosine 5′-monophosphate-activated protein kinase,AMPK)/细胞外调节蛋白激酶(extracellular regulated protein kinases,ERK)/雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)自噬信号途径的影响。方法:TCID 50检测DENV2对C6/36细胞的毒力,RT-PCR检测DENV2感染后HUVEC内病毒NS1基因部分片段;透射电子显微镜观察自噬体结构,Western blot检测不同剂量病毒感染HUVEC后对微管关联蛋白Ⅱ(microtubule-associated proteinsⅡ,LC3-Ⅱ)表达的影响和不同时间MIF的蛋白质水平变化,分析MIF与自噬相关蛋白LC3-Ⅱ的相关性;MIF抑制剂和通路抑制剂分别预处理DENV2感染的HUVEC,Western blot检测MIF、AMPK、ERK、mTOR和LC3-Ⅱ蛋白变化,分析MIF与AMPK自噬通路的关联。结果:实验用毒株对C6/36细胞的TCID 50为10-9.09/ml,被感染的HUVEC内可检测病毒NS1基因部分片段序列。不同剂量病毒感染后,在HUVEC内可形成自噬小体或自噬溶酶体,自噬水平有差异。DENV2感染HUVEC引起MIF和LC3-ⅡmRNA水平的变化呈正相关,MIF抑制剂的处理影响通路蛋白AMPK、ERK和LC3-Ⅱ的表达,但几乎不影响MIF的蛋白质水平。结论:DENV2感染HUVEC引起的MIF变化可影响自噬水平,MIF通过调控AMPK/ERK/mTOR途径影响自噬。
Objective To investigate the effects of macrophage migration inhibitory factor(MIF)on AMPK/ERK/mTOR autophagy signaling pathway in primary human umbilical vein endothelial cells(HUVEC)after dengue virus type 2(DENV2)infection.Methods The virulence of DENV2 to C6/36 cells was assessed with 50%tissue culture infectious dose(TCID50).NS1 gene fragments in DENV2-infected HUVEC were detected by RT-PCR.Transmission electron microscopy was used to detect autophagosomes.Western blot was performed to detect the effects of DENV2 infection on the expression of autophagy-related protein LC3-Ⅱand MIF in HUVEC.The correction of MIF with LC3-Ⅱwas then analyzed.HUVEC were pretreated with MIF inhibitor(ISO-1)or pathway inhibitor(Compound C or U0126),and then the changes in the expression of MIF,adenosine 5′-monophosphate-activated protein kinase(AMPK)pathway-related proteins and LC3-Ⅱafter DENV2 infection were detected by Western blot to reveal the correlation between MIF and AMPK autophagy pathway.Results The TCID50 to C6/36 cells was 10-9.09/ml in this experiment.NS1 gene fragments were detected in DENV2-infected HUVEC.Autophagosomes or autophagolysosomes were observed in the infected HUVEC and there were differences in autophagy induced by different doses of DENV2.The mRNA levels of MIF and LC3-Ⅱin HUVEC were positively correlated after DENV2 infection.MIF inhibitor affected AMPK,ERK and LC3-Ⅱlevels,but had no significant influence on MIF expression at protein level.Conclusions MIF could affect autophagy through regulating the AMPK/ERK/mTOR signaling pathway in HUVEC during DENV2 infection.
作者
苟小琴
吴宁
左丽
Gou Xiaoqin;Wu Ning;Zuo Li(Department of Immunology,College of Basic Medicine,Guizhou Medical University,Guiyang 550000,China)
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2022年第3期185-193,共9页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金(81860289)。
