^(60)Co γ线照射血管内皮细胞外泌体中miRNA的鉴定分析  

Identification and analysis of exosomal miRNAs in human umbilical vein endothelial cells irradiated by ^(60)Co γ-rays

作　　者：黄巧华[1] 宋曼[2] 高山山[2] 莫丽君[1] 刘晓丹 王豫[2] 黄波[1] 周平坤[2] 

机构地区：[1]南华大学公共卫生学院,湖南衡阳421001 [2]军事医学科学院放射与辐射医学研究所北京市放射生物学重点实验室,北京100850

出　　处：《军事医学》2017年第5期367-372,共6页Military Medical Sciences

基　　金：国家自然科学基金重点资助项目(81530085);湖南省自然科学基金资助项目(2016JJ2115)

摘　　要：目的利用微小RNA(miRNA)芯片和生物信息学技术,研究受照射人脐静脉血管内皮细胞(HUVEC)产生的外泌体miRNA组分的变化,为揭示血管组织放射损伤及其旁效应机制提供新的线索。方法超高速离心法收集对照组和4 Gy剂量照射组的HUVEC外泌体,运用电镜及Western印迹技术对外泌体进行鉴定,miRNA芯片技术分析细胞内和外泌体中miRNA表达谱,qRT-PCR法验证部分差异miRNA,通过miRDB和Target Scan预测差异miRNA的靶基因,DAVID、KEGG等在线工具进行生物信息学分析。结果 HUVEC经4 Gy照射后外泌体miRNA与对照组相比,照后0.5 h共鉴定到18个发生表达变化的miRNA分子,5个表达上调,13个表达下调;照后2 h鉴定出16个表达上调、5个表达下调miRNA分子;细胞内miRNA与对照组相比,照射后0.5、2 h分别有38个和85个差异表达miRNA,且差异有统计学意义(P<0.01)。生物信息学结果表明,这些表达变化的miRNA可能通过参与调控MAPK、Ras、PI3K-Akt信号通路等途径影响细胞辐射旁效应。结论电离辐射损伤导致血管内皮细胞外泌体miRNA分子组分和表达水平发生显著改变,这些miRNA的靶基因组产物在细胞放射损伤反应的信号通路调节中发挥重要作用。Objective To study the changes in miRNAs expression in the exosomes of human umbilical vein endothelial cells（HUVECs） after 60Co γ-rays expose using microRNA（miRNA） chips and bioinformatics techniques so as to provide new clues to the mechanism of radiation-induced vascular tissue injury and its bystander effects. Methods HUVECs exosomes were collected in the control and 4 Gy irradiated cells by ultra-high-speed centrifugation, and further confirmed using transmission electron microscopy （TEM） and Western blotting of exosomes biomarkers, miRNA microarray was used to analyze miRNA expression profiles of exosomes and cells. Also, real-time quantitative PCR （qRT-PCR） was used to verify differentially expressed miRNAs, and the miRDB and TargetScan were performed to predict the target genes of the differentially expressed miRNAs. Bioinformatics analysis was performed using DAVID, KEGG and other online tools. Results Compared with the control exosomes from non-irradiated HUVECs, miRNA microarray analysis revealed that 5 up-regulated, and 13 down-regulated miRNAs were identified in the exosomes from HUVECs at 0.5 h after 4 Gy-irradiation, and 16 up-regulated and 5 down-regulated miRNAs at 2 h after 4 Gy-irradiation. Moreover, 38 and 85 miRNAs were differentially expressed respectively in the HUVECs at 0.5 h and 2 h after radiation. The difference was statistically significant（ P 〈 0.01 ）. The results of bioinformaties showed that these miRNAs might exert the radiation-induced bystander effect （RIBE） by regulating MAPK signal pathways, RAS and PI3K-Akt signal pathways. Conclusion The ionizingradiation injury significantly alters the components and expression levels of exosomal miRNAs, which play important roles in regulating the signal pathways in response to radiation.

关 键 词：Γ射线 内皮 血管 外泌体 微小RNA 芯片分析技术 生物信息学 

分 类 号：R818[医药卫生—放射医学]