热打击诱导血管内皮细胞释放的外泌体特征及其功能分析

Features and function of exosomes secreted by heat-stroke vascular endothelial cells

目的分析热打击诱导人脐静脉内皮细胞(HUVECs)释放的外泌体特征及其功能。方法培养HUVECs并分为对照组与热打击组。热打击组细胞给予41℃热刺激培养2 h,建立热打击细胞模型;对照组细胞于37℃孵育培养2 h。采用纳米颗粒追踪法分析两组细胞释放的外泌体特征;Westernblotting检测外泌体标志蛋白CD63和TSG101的表达;荧光标识观察外泌体进入正常细胞的过程。检测、提取外泌体,对细胞进行干预实验,将培养的细胞分为空白对照组(37℃培养HUVECs细胞)、37℃外泌体处理组(加入对照组细胞释放的外泌体)和41℃外泌体处理组(加入热打击组细胞释放的外泌体),采用流式细胞仪检测细胞周期和细胞凋亡情况;MTS法检测细胞增殖情况;ELISA法检测凝血功能指标水平;高通量测序法分析外泌体中的微小RNA(miRNA)。结果与对照组不同,41℃热打击组细胞可释放异常外泌体,并导致正常HUVECs出现行为、功能异常,组织因子、血管性血友病因子、纤溶酶原激活物抑制因子1表达明显增加(P<0.05),组织型纤维蛋白溶酶原激活剂表达降低(P<0.05)。与37℃孵育HUVECs比较,41℃热打击HUVECs释放的外泌体共出现2590条miRNA调节变化,对细胞凋亡等途径具有较明显影响。结论41℃热打击可使HUVECs分泌异常外泌体,后者可导致正常细胞出现行为及凝血功能等的异常。

Objective To characterize exosomes released from human umbilical vein endothelial cells(HUVECs)after heatstroke,and explore their effects on biological behaviors of cells.Methods HUVECs were cultured and divided into the control group and the heat shock group.The heat shock group cells were exposed at 41℃for 2 hours;the control group cells were incubated at 37℃for 2 hours.The characteristics of exosomes secreted by the two groups were analyzed by nanoparticle tracing.The expression of exosomes marker proteins,CD63 and TSG101,were detected by Western blotting.The process of exosomes entering normal cells was observed by fluorescence labeling.To further explore the potential function of exosomes secreted from the heat-shocked cells,cells were co-cultured with exosomes collected from cells cultured at 37℃or cells cultured at 41℃.Cells under normal culture conditions were served as a negative control(blank control).FACS was used to detect cell cycle and apoptosis;MTS was used to evaluate cell proliferation;ELISA was used to quantify coagulation related factor expression;high-throughput sequencing was used to profile microRNAs(miRNAs)in the exosomes.Results Different from the control group,the cells in 41℃heat shock group secreted abnormal exosomes,which resulted in abnormal behaviors and functions of normal HUVECs.The expression of tissue factor,von Willebrand factor,plasminogen activator inhibitor-1 increased significantly(P<0.05),and the expression of tissue plasminogen activator decreased(P<0.05).Compared with the incubation of HUVECs at 37℃,there were 2590 miRNA regulatory changes in exosomes secreted by HUVECs after heat shock at 41℃,which had obvious effects on apoptosis.Conclusions Heat shock at 41℃could induce the release of abnormal exosomes from HUVEC,leading to abnormal behavior and potential coagulation of normal cells.