脓毒症外泌体miR-1306-5p对肠黏膜上皮细胞炎症、凋亡和氧化应激的影响  

Effects of exosomal miR-1306-5p in sepsis on inflammation,apoptosis,and oxidative stress in intestinal mucosal epithelial cells

作　　者：宋婷婷[1] 柴瑞峰[1] 李颖[1] 史星宇 李建[1] SONG Tingting;CHAI Ruifeng;LI Ying;SHI Xingyu;LI Jian(Department of Critical Care Medicine,First Affiliated Hospital of Xinjiang Medical University,Urumqi,Xinjiang 830054,China)

机构地区：[1]新疆医科大学第一附属医院重症医学科,新疆乌鲁木齐830054

出　　处：《国际检验医学杂志》2024年第20期2449-2455,共7页International Journal of Laboratory Medicine

基　　金：新疆维吾尔自治区自然科学基金项目(2021D01C306)。

摘　　要：目的研究脓毒症血浆源性外泌体微小RNA-1306-5p(miR-1306-5p)对肠黏膜上皮细胞的炎症、凋亡和氧化应激损伤的调控作用,并探讨潜在机制。方法分离脓毒症血浆源性外泌体和健康血浆外泌体,分为健康血浆外泌体组和脓毒症血浆源性外泌体组。用电镜观察外泌体,分析两组外泌体物理参数,并检测外泌体中miR-1306-5p的表达。将肠黏膜上皮细胞分为对照组、miR-1306-5p模拟物的阴性对照组(mimic-NC组)、miR-1306-5p模拟物组(mimic组)、mimic联合过表达Polo样激酶1(PLK1)的空载体组(mimic-PLK1-EV组)、mimic联合过表达PLK1组(mimic+PLK1-OE组)。采用实时荧光定量PCR检测各组中miR-1306-5p和PLK1 mRNA的表达,蛋白质印记法检测miR-1306-5p的靶基因PLK1、胱天蛋白酶(caspase)3、B淋巴细胞瘤-2(Bcl-2)、Bcl2-相关X蛋白(Bax)的表达,双荧光素酶报告基因法检测miR-1306-5p和PLK1的结合作用,采用流式细胞术检测细胞凋亡,酶联免疫吸附试验检测培养细胞的上清液中肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-6、IL-8的表达,试剂盒法检测细胞中活性氧(ROS)、丙二醛(MDA)、谷胱甘肽(GSH)及超氧化物歧化酶(SOD)的表达。结果脓毒症血浆源性外泌体和健康血浆外泌体均呈椭球形,物理参数之间的差异无统计学意义(P>0.05),与健康血浆外泌体组比较,脓毒症血浆源性外泌体组中的miR-1306-5p的表达上调(P<0.05)。双荧光素酶报告基因法证实,PLK1是miR-1306-5p的靶基因。与mimic-NC组比较,mimic组miR-1306-5p、TNF-α、IL-6、IL-8、IL-1β、caspase3、Bax、ROS、MDA的表达上调,细胞凋亡率增加,PKL1、Bcl-2、GSH、SOD的表达下调,差异均有统计学意义(P<0.05)。与mimic+PLK1-EV组比较,mimic+PLK1-OE组TNF-α、IL-6、IL-8、IL-1β、caspase3、Bax、ROS、MDA的表达明显下调,细胞凋亡率降低,PKL1、Bcl-2、GSH、SOD的表达上调,差异均有统计学意义(P<0.05)。结论脓毒症血浆源性外泌体miR-1306-5Objective To investigate the regulatory effects of plasma-derived exosomal microRNA-1306-5p(miR-1306-5p)on inflammation,apoptosis and oxidative stress in intestinal mucosal epithelial cells in sepsis,and to explore the potential mechanisms.Methods Sepsis plasma-derived exosomes and healthy plasma exosomes were separated and divided into healthy plasma exosomes group and sepsis plasma-derived exosomes group.The exosomes were observed by electron microscopy,the physical parameters of the two groups of exosomes were analyzed,and the expression of miR-1306-5p in the exosomes was detected.Intestinal mucosal epithelial cells were divided into control group,negative control group of miR-1306-5p mimic(mimic-NC group),miR-1306-5p mimic group(mimic group),mimic combined with overexpression of PLK1 empty vector group(mimic-PLK1-EV group),and mimic combined with overexpression of Polo-like kinase 1(PLK1)group(mimic+PLK1-OE group).Real-time fluorescent quantitative PCR was used to detect the mRNA expressions of miR-1306-5p and PLK1 in each group,and protein imprinting was used to detect the expressions of miR-1306-5p target genes PLK1,caspase 3,B lymphoblastoma-2(Bcl-2)and Bcl2-associated X protein(Bax).Dual luciferase reporter gene assay was used to detect the binding effect of miR-1306-5p and PLK1,and flow cytometry was used to detect apoptosis.The expressions of tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,IL-6 and IL-8 in the supernatant of cultured cells were detected by enzyme-linked immunosorbent assay.The expressions of reactive oxygen species(ROS),malondialdehyde(MDA),glutathione(GSH)and superoxide dismutase(SOD)were detected by kit method.Results The plasma derived exosomes of sepsis and healthy plasma were ellipsoid in shape,and there was no significant difference in physical parameters(P>0.05).Compared with the healthy plasma exosomes group,the expression of miR-1306-5p was up-regulated in the plasma derived exosomes of sepsis group(P<0.05).PLK1 was identified as the target gene of miR-1306-5p by double lu

关 键 词：脓毒症 外泌体 微小RNA-1306-5p 肠黏膜上皮细胞 

分 类 号：R446.1[医药卫生—诊断学]