内质网应激肝癌细胞通过外泌体-Toll样受体4信号途径促进巨噬细胞M2极化  

Endoplasmic reticulum stressed hepatocellular carcinoma promotes macrophage M2 polarization through exosome-toll-like receptor 4 pathway

作　　者：化维 朱嫚嫚 范璐璐[2] 孙国平[2] 刘加涛[1] HUA Wei;ZHU Man-man;FAN Lu-lu;SUN Guo-ping;LIU Jia-tao(Dept of Pharmacy,the First Affiliated Hospital of Anhui Medical University,Hefei 230022,China;Dept of Oncology,the First Affiliated Hospital of Anhui Medical University,Hefei 230022,China)

机构地区：[1]安徽医科大学第一附属医院药剂科,安徽合肥230022 [2]安徽医科大学第一附属医院肿瘤科,安徽合肥230022

出　　处：《中国药理学通报》2021年第7期1008-1015,共8页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 82072687);安徽省自然科学基金(No 2008085MH257);高校优秀青年人才支持计划(No gxyq2020008)。

摘　　要：目的探讨肝癌细胞内质网应激相关外泌体能否通过Toll样受体4(TLR4)调控巨噬细胞M2极化及潜在机制。方法采用Western blot实验分析肝癌组织和衣霉素刺激后肝癌细胞内质网应激相关标志蛋白GRP78的表达及肝癌细胞分泌的外泌体鉴定;免疫组化检测肝癌组织GRP78的表达水平;免疫荧光验证巨噬细胞有效摄取外泌体;流式细胞术、Western blot实验和CBA试剂盒检测巨噬细胞M2型表型转化相关标志分子及巨噬细胞Toll样受体4表达水平;通过Affymetrix基因芯片和生物信息学分析筛选内质网应激相关外泌体作用后巨噬细胞mRNA的差异表达及TLR4的表达。结果临床样本中,肝癌组织GRP78表达高于癌旁组织。在细胞实验中,与对照组相比,2.5μmol·L^(-1)的衣霉素刺激肝癌细胞24 h可明显升高内质网应激标志蛋白GRP78表达。衣霉素刺激和未刺激的肝癌细胞分泌的外泌体均特异性表达CD63、TSG101、HSP70外泌体标志性蛋白,但不表达外泌体阴性蛋白Calnexin。处理组肝癌细胞分泌的外泌体中HSP70表达高于对照组。激光共聚焦显微镜提示巨噬细胞可有效摄取外泌体,流式细胞术和Western blot进一步提示肝癌细胞ERS相关外泌体可显著上调巨噬细胞CD206、转化生长因子β1(TGF-β1)和精氨酸酶1(Arg-1)的表达。此外,这些外泌体可以促进巨噬细胞分泌IL-6和IL-10,并上调巨噬细胞TLR4表达。结论内质网应激的HCC细胞可能通过释放富含HSP70的外泌体,激活TLR4信号,促进巨噬细胞M2极化。Aim To explore the effect of endoplasmic reticulum(ER)stress-related exosomes derived from hepatocellular carcinoma(HCC)cells on the M2 polarization of macrophages via Toll-like receptor 4(TLR4)and the underlying mechanism.Methods Western blot experiments were conducted to evaluate the GRP78 levels in HCC tissues and HCC cells with or without pre-treatment of tunicamycin and the identification of the isolated exosomes.Immunohistochemical staining was also employed to detect the expression of GRP78 in HCC tissues.Immunofluorescence was used to confirm the effective absorbance of exosomes by macrophages.Flow cytometry(FCM),Western blot and CBA kit were used to detect M2 polarization-associated markers and the expression of TLR4 on macrophages.Affymetrix gene chip and bioinformatics analysis were used to screen the differential expression of mRNA in macrophages stimulated by ER stressed-related exosomes and TLR4 expression.Results In clinical samples,GRP78 expression in liver cancer tissues was higher than that in the adjacent tissues.In cell experiments,tunicamycin(2.5μmol·L^(-1))treatment for 24 hours in HCC cells could significantly increase the expression of ER stress marker protein GRP78 compared with control group.Western blot showed that HCC cell-derived exosomes positively expressed exosomal marker proteins,such as CD63,TSG101 and HSP70,and negatively expressed Calnexin.Interestingly,HSP70-enriched exosomes released by ER-stressed HCC cells were observed.Laser confocal microscopy found that exosomes could be effectively taken up by RAW264.7 macrophages.Flow cytometry and Western blot further indicated that ER stress-related exosomes could significantly up-regulate the expression of CD206,transformed growth factor-beta(TGF-β)and arginase-1(Arg-1)in macrophages.Meanwhile,these exosomes could promote the secretion of IL-6 and IL-10,and up-regulate the expression of TLR4 in macrophages.Conclusions ER stressed-HCC cells could promote macrophage M2 polarization by releasing HSP70-enriched exosomes and activatin

关 键 词：肝细胞癌 内质网应激 巨噬细胞 外泌体 TOLL样受体4 极化 

分 类 号：R329.24[医药卫生—人体解剖和组织胚胎学] R392.11[医药卫生—基础医学]