骨髓间充质干细胞源外泌体对大鼠颈动脉球囊损伤后circRNA和microRNA差异表达的影响  

Effects of exosomes derived from bone marrow mesenchymal stem cells on the differential expression of circRNA and microRNA after balloon injury of rat carotid artery

作　　者：周涵 邓文文 王冬梅 龙仙萍 蒋礼祥 杨双亚 郭鸿浩 陈文明 石蓓 ZHOU Han;DENG Wen-wen;WANG Dong-mei;LONG Xian-ping;JIANG Li-xiang;YANG Shuang-ya;GUO Hong-hao;CHEN Wen-ming;SHI Bei(Department of Cardiology,Affiliated Hospital of Zunyi Medical University,Zunyi 563000,Guizhou,China)

机构地区：[1]遵义医科大学附属医院心内科,遵义563000

出　　处：《医学研究生学报》2021年第4期341-349,共9页Journal of Medical Postgraduates

基　　金：国家自然科学基金(81560041,81860063)。

摘　　要：目的骨髓间充质干细胞(BMSCs)能够在多种心血管疾病病理过程中发挥重要作用,但其对支架内再狭窄的影响有待进一步研究。文中旨在探讨BMSCs源外泌体对大鼠颈动脉球囊损伤后新生内膜增殖的影响及circRNA和microRNA表达变化。方法采用差速超速离心法提取大鼠BMSCs源外泌体,并使用Western blot及透射电镜对其进行鉴定。采用球囊损伤大鼠颈动脉的方法建立血管内膜增生动物模型,将36只成年雄性SD大鼠随机分为:假手术组、损伤组、外泌体组(400μL);假手术组大鼠只分离颈左总动脉血管,其余各组大鼠的左颈总动脉均行球囊损伤,外泌体组行球囊损伤后立即原位注射400μL BMSCs源外泌体并用微型血管夹夹闭血管5 min。采用HE染色检测内膜增生情况,DCFH-DA染色检测血管活性氧(ROS)释放情况。采用circRNA和microRNA芯片筛查差异表达的circRNA和microRNA,并以每组3个样本对其中显著差异的8个circRNA和7个microRNA进行qRT-PCR验证,并筛选出BMSCs源外泌体处理后差异表达的circRNA和microRNA;生物信息学方法预测差异microRNA的靶基因,并进行GO分析和KEGG通路分析,构建circRNA-microRNA-mRNA共表达网络,对其潜在分子机制进行探讨。结果透射电镜及Western blot验证提取的囊泡状物质为外泌体;HE染色结果显示:损伤组较假手术组新生内膜增殖明显,其内膜/中膜面积比(1.902±0.217)较假手术组(1.000±0.000)显著增加(P<0.05);外泌体组较损伤组新生内膜增殖程度有所减低,其内膜/中膜面积比(0.982±0.067)较损伤组明显减低(P<0.05)。DCFH-DA染色结果显示:与假手术组DCFHA红色荧光强度(4.730±0.104)相比,损伤组(16.800±0.380)明显增强(P<0.05);与损伤组相比,外泌体组DCFHA红色荧光强度(12.810±0.094)明显减弱(P<0.05)。circRNA芯片显示差异性表达的circRNA共1683个(Fold change≥2,P<0.05)。与假手术组相比,损伤组中上调的有631个,下调的有1052个。micObjective Bone marrow mesenchymal stem cells(BMSCs)play an important role in a variety of cardiovascular diseases.However,its effect on in-stent restenosis remains to be further explored.To explore the effect of BMSC-derived exosomes on the neointimal proliferation and the expression changes of circRNA and microRNA after carotid artery balloon injury in rats.Methods Rat BMSCs exocrine was extracted by differential ultracentrifugation and identified by Western blot(WB)and transmission electron microscope.An animal model of vascular intimal hyperplasia was established by ballooning the carotid artery of rats.A total of 36 adult male SD rats were randomly divided into:①sham operation group,②injury group,③exosomes group(400μL).HE staining was used to detect intimal hyperplasia,and DCFH-DA staining was used to detect vascular reactive oxygen species(ROS)release.The circRNA and microRNA expression profile chip screening in the control group and the rat carotid artery in carotid artery balloon injury group organization differentially expressed circRNA and microRNA.With 3 samples in each group,the 8 circRNAs and 7 microRNAs with significant differences were verified by qRT-PCR,and the differentially expressed circRNA and microRNAs after treatment with BMSCs-derived exosomes were screened out.At the same time,bioinformatics analyses including circRNA-microRNA and microRNA-mRNA networks were constructed and GO and KEGG Pathway analyses were performed for the top 10 circRNAs.Results Transmission electron microscopy and WB verify that the extracted vesicles are exosomes.Compared with the injury group,BMSCs-derived exosomes significantly inhibited the degree of intimal hyperplasia(P<0.05)and the content of intravascular ROS(P<0.05).circRNA microarrays showed that 1683 circRNAs were differentially expressed between the carotid artery balloon injury group and the control group(Fold change≥2,P<0.05).In all,631 of them were significantly up-regulated,and the other 1052 were down-regulated in the carotid artery balloon in

关 键 词：外泌体 骨髓间充质干细胞 circRNA MICRORNA 颈动脉球囊损伤模型 

分 类 号：R543.5[医药卫生—心血管疾病]