脂肪间充质干细胞来源外泌体对阿霉素所致心肌损伤的影响及其分子机制  

Effect and mechanism of adipose-derived mesenchymal stem cell-derived exosomes on myocardial injury induced by adriamycin

作　　者：陈睿 马骞 傅建芳 张婧 朱军 王伟东 王旁 CHEN Rui;MA Qian;FU Jianfang;ZHANG Jing;ZHU Jun;WANG Weidong;WANG Pang(Xijing Hospital,Airforce Military Medical University,Xi’an 710032,China)

机构地区：[1]空军军医大学西京医院,西安710032

出　　处：《山东医药》2021年第11期15-19,23,共6页Shandong Medical Journal

基　　金：陕西省重点研发计划一般项目(2020SF-322)。

摘　　要：目的观察小鼠脂肪间充质干细胞(mADSC)来源外泌体对阿霉素所致心肌损伤的影响,并探讨其可能的机制。方法通过外泌体提取试剂盒Exo-quick提取雄性C57小鼠的mADSC来源外泌体及血浆外泌体,并通过透射电镜、Western blotting法以及粒径分析技术进行鉴定证实。将H9C2细胞分为损伤1组(加入阿霉素)、处理1组(加入阿霉素和2μg/mL的mADSC来源外泌体)和对照1组(加入PBS),TUNEL染色后对凋亡细胞进行计数,流式细胞术检测细胞凋亡率。取雄性C57小鼠18只,随机分为损伤2组(尾静脉注射阿霉素)、处理2组(尾静脉注射阿霉素和mADSC来源外泌体)和对照2组(尾静脉注射等体积生理盐水),每组6只,连续给药4周;部分小鼠处死后取心肌组织,TUNEL染色后对凋亡细胞进行计数,流式细胞术检测细胞凋亡率;部分小鼠行M型超声心动图检查,测量左室射血分数(LVEF)。采用实时荧光定量PCR法检测小鼠血浆外泌体与mADSC来源外泌体中miR-21a-5p表达,并进行miR-21a-5p作用靶点的基因本体(GO)功能和京都基因与基因组百科全书(KEGG)通路富集分析。结果损伤1组、处理1组和对照1组细胞凋亡数和细胞凋亡率均逐渐降低,组间两两比较P均<0.05。损伤2组、处理2组和对照2组小鼠心肌组织细胞凋亡数、细胞凋亡率、纤维化比例均依次降低,LVEF依次升高,组间两两比较P均<0.05。小鼠血浆外泌体与mADSC来源外泌体中miR-21a-5p相对表达量分别为1.05±0.01、5.41±0.05,二者比较P<0.05。GO功能、KEGG通路富集分析结果显示,miR-21a-5p的调控通路包括DNA损伤修复、mTOR信号通路、血管平滑肌舒张相关信号通路、脂肪酸代谢相关通路等。结论 mADSC来源外泌体可减少阿霉素所致的心肌细胞凋亡,减轻阿霉素所致小鼠心肌纤维化并提高其心功能,其机制可能与升高miR-21a-5p表达进而调控多种信号通路有关。Objective To explore the protective effect of exosomes derived from mouse adipose-derived mesenchymal stem cells(mADSC) on doxorubicin(Dox) induced myocardial injury.Methods The exosomes derived from mADSC were extracted by Exo-quick exosome extraction kit,and the exosomes derived from mADSC were identified by transmission electron microscopy(TEM),Western blotting and particle size analysis(NTA).H9 c2 cells were divided into Dox1 group(added with doxorubicin),Dox+mADSC-Exo 1 group(added with doxorubicin and 2 μg/mL of madsc derived exosomes),and control 1 group(added with PBS),and apoptotic cells were counted after TUNEL staining and the apoptosis rate was determined by flow cytometry.A total of 18 male C57 mice were randomly divided into three groups:Dox 2 group(injected with doxorubicin vin tail vein),Dox+mADSC-Exo 2 group(injected with doxorubicin and madsc derived exosomes via tail vein),and control 2 group(injected with an equal volume of saline via tail vein),with 6 in each group,and were given corresponding drugs for 4 weeks.A part of mice were sacrificed,and myocardial tissues were taken,apoptotic cells were counted after TUNEL staining and the apoptosis rate was determined by flow cytometry;a part of mice underwent M-mode echocardiography,and left ventricular ejection fraction(LVEF) was measured.Real-time fluorescence quantification PCR was used to detect the expression of miR-21 a-5 p in mice plasma exosomes and mADSC derived exosomes,and gene ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG) functional enrichment analysis of miR-21 a-5 p targets was performed.The targets of miR-21 a-5 p were analyzed by gene ontology(GO) function and Kyoto Encyclopedia of genes genomes(KEGG) pathway enrichment.Results The number of apoptotic cells and apoptotic rate in Dox 1 group,Dox+mADSC-Exo 1 group and control 1 group decreased gradually(all P<0.05).The number of apoptotic cells,apoptosis rate,and fibrosis ratio in the myocardium of the mice in the Dox 2 group,Dox+mADSC-Exo 2 group,and control 2 group dec

关 键 词：脂肪间充质干细胞 外泌体 阿霉素 心肌损伤 miR-21a-5p 小鼠 

分 类 号：R542.2[医药卫生—心血管疾病]