缺氧肝癌源性外泌体miR-1260b对肿瘤相关巨噬细胞M2亚型的影响及其机制  

EFFECTS OF HYPOXIC HEPATOMA-DERIVED EXOSOME miR-1260b ON M2 MACROPHAGES AND MECHANISM

作　　者：杨晔妮 赵梓吟 王有鹏 孙洪发 张顺[1] 韩冰[1] YANG Yeni;ZHAO Ziyin;WANG Youpeng;SUN Hongfa;ZHANG Shun;HAN Bing(Department of Hepatobiliary Surgery,The Affiliated Hospital of Qingdao University,Qingdao 266003,China)

机构地区：[1]青岛大学附属医院肝胆胰外科,山东青岛266003 [2]青岛大学附属医院器官移植中心,山东青岛266003

出　　处：《精准医学杂志》2023年第2期105-110,115,共7页Journal of Precision Medicine

摘　　要：目的探究缺氧肝癌源性外泌体miR-1260b对肿瘤相关巨噬细胞M2亚型的影响及其机制。方法人肝癌细胞97H(MHCC97H)细胞经100μmol/L CoCl 2处理24 h后获得缺氧97H细胞。利用丙二醇甲醚醋酸酯(PMA)处理THP-1细胞24 h后获得M0巨噬细胞,实时荧光定量PCR(RT-qPCR)方法检测THP-1细胞中CD 11b的相对表达量。将常氧/缺氧97H细胞与M0巨噬细胞共培养48 h后,采用RT-qPCR方法检测巨噬细胞中CD 163、CD 206和TNF-α基因相对表达量。通过低温差速离心法提取常氧/缺氧97H细胞的外泌体,通过生物透射电子显微镜观察外泌体的形态,采用纳米颗粒跟踪分析仪(NTA)分析粒子直径,采用Western blot法检测外泌体中凋亡诱导因子6相互作用蛋白(Alix)和钙联蛋白相对表达量。外泌体与M0巨噬细胞共培养48 h后,采用RT-qPCR方法检测M0巨噬细胞中CD 206相对表达量。用携带Dil染剂的外泌体与M0巨噬细胞共培养24 h,采用荧光显微镜观察M0巨噬细胞内的荧光反应。采用RT-qPCR方法检测外泌体中miR-1260b相对表达量,并向M0、M2巨噬细胞内转染miR-1260b Mimic/Inhibitor后,采用RT-qPCR方法检测细胞内CD 206、肿瘤坏死因子-α(TNF-α)相对表达量。将转染了miR-1260b Inhibitor的常氧/缺氧97H细胞共培养M0巨噬细胞,采用RT-qPCR方法检测M0巨噬细胞CD 206、TNF-α相对表达量。结果PMA处理THP-1细胞24 h后所获得的M0巨噬细胞CD 11b显著高表达(t=78.14,P<0.05)。缺氧97H细胞与M0巨噬细胞共培养可以使巨噬细胞CD 163以及CD 206的表达升高,TNF-α的表达降低(t=14.23~46.88,P<0.05)。成功分离并提取缺氧97H细胞分泌的外泌体颗粒,其可以被M0巨噬细胞吞噬。Western blot结果显示,外泌体颗粒高表达Alix蛋白且基本不表达钙联蛋白。RT-qPCR检测显示,缺氧肝癌源性外泌体共培养M0巨噬细胞后可以使M0巨噬细胞高表达CD 206(t=17.06,P<0.05);缺氧肝癌源性外泌体高表达miR-1260b(t=12.09,P<0.05);转染miR-1260b MimObjective To explore the effects of the hypoxic hepatoma-derived exosome miR-1260b on the M2 subtype of tumor-associated macrophages and the underlying mechanism.Methods MHCC97H(97H)cells were treated with 100μmol/L CoCl 2 for 24 h to obtain hypoxic 97H cells.THP-1 cells were treated with propylene glycol methyl ether acetate(PMA)for 24 h to derive M0 macrophages.The relative expression of CD 11b in THP-1 cells was measured by real-time quantitative PCR(RT-qPCR).After co-culture with normoxic/hypoxic 97H cells for 48 h,the relative gene expression of CD163,CD206,and TNF-αin M0 macrophages was measured by RT-qPCR.The exosomes of normoxic/hypoxic 97H cells were extracted by low temperature differential centrifugation.A transmission electron microscope was used to observe the shape of the exosomes.The particle diameter was measured using a nanoparticle tracking analyzer.Western blot was used to determine the relative expression of apoptosis-inducing factor 6-interacting protein(Alix)and calnexin in the exosomes.After co-culture with the exosomes for 48 h,the relative expression of CD206 in M0 macrophages was measured by RT-qPCR.The exosomes labeled with Dil stain were co-cultured with M0 macrophages for 24 h,and then M0 macrophages were examined for the fluorescence reaction using a fluorescence microscope.The expression of miR-1260b in the exosomes was measured by RT-qPCR.After M0 and M2 macrophages were transfected with miR-1260b Mimic/Inhibitor,the expression of CD206 and TNF-αin the cells was determined by RT-qPCR.After co-culture with normoxic/hypoxic 97H cells transfected with miR-1260b Inhibitor,M0 macrophages were measured for the relative expression of CD206 and TNF-αby RT-qPCR.Results There was significantly high expression of CD 11b in M0 macrophages derived from THP-1 cells after 24 h PMA treatment(t=78.14,P<0.05).M0 macrophages co-cultured with hypoxic 97H cells showed significantly increased expression of CD163 and CD206 and significantly decreased expression of TNF-α(t=14.23-46.88,P<0.05).The exo

关 键 词：肝肿瘤 细胞低氧 外泌体 微RNAS 转染 巨噬细胞 肿瘤逃逸 

分 类 号：R735.7[医药卫生—肿瘤]