脂肪间充质干细胞来源外泌体miR-126-3p对人脐静脉内皮细胞糖脂毒性的作用  

Effects of miR-126-3p from adipose-derived mesenchymal stem cell released exosomes on human umbilical vein endothelial cell glucolipotoxicity

作　　者：林波 陈鑫宇 金秋 朱芝漫 赵文慧 Lin Bo;Chen Xinyu;Jin Qiu;Zhu Zhiman;Zhao Wenhui(Basic Medical Department,Jiangsu Vocational College of Nursing,Huaian 223005,Jiangsu Province,China)

机构地区：[1]江苏护理职业学院医学基础部,江苏省淮安市223005

出　　处：《中国组织工程研究》2022年第30期4773-4779,共7页Chinese Journal of Tissue Engineering Research

基　　金：江西省教育厅科技项目(GJJ201243),项目参与人:林波。

摘　　要：背景:脂肪间充质干细胞衍生外泌体(adipose-derived mesenchymal stem cells released exosomes,AMSC-Exos)中miR-126-3p可作为糖尿病的一种潜在生物标志物,但其对人脐静脉内皮细胞糖脂毒性的作用及其相关机制研究较少。目的:探究AMSC-Exos中miR-126-3p对人脐静脉内皮细胞糖脂毒性的效用及作用机制。方法:①以30 mmol/L葡萄糖与100μmol/L棕榈酸诱导人脐静脉内皮细胞糖脂毒性体外模型;②收集第4代脂肪间充质干细胞上清液提取外泌体,透射电镜观察其形态,纳米颗粒跟踪分析外泌体粒径分布范围;③AMSC-Exos处理糖脂毒性人脐静脉内皮细胞,流式细胞术检测活性氧水平及细胞凋亡率,ELISA检测培养上清中炎症因子水平;④RT-qPCR分析AMSC-Exos、与脂肪间充质干细胞共培养后正常人脐静脉内皮细胞中miR-126-3p mRNA相对表达水平;⑤miR-126-3p转染糖脂毒性人脐静脉内皮细胞,CCK-8检测细胞增殖情况,Western blot检测凋亡蛋白的表达量;⑥生物信息学软件预测和分析miR-126-3p靶基因,双荧光素酶报告基因实验进行验证,并预测信号通路;⑦CRK转染糖脂毒性人脐静脉内皮细胞,Western blot检测凋亡蛋白及p-AKT的表达量;⑧AKT通路抑制剂RG7440与miR-126-3p过表达/AMSC-Exos共处理糖脂毒性人脐静脉内皮细胞,Western blot检测凋亡蛋白的表达量,流式细胞术检测细胞凋亡率。结果与结论:①AMSC-Exos为形态一致的圆形或椭圆形膜性囊泡,粒径范围30-200 nm;②与模型组相比,AMSC-Exos可明显降低白细胞介素1β、白细胞介素6和白细胞介素8的表达量(P<0.05,P<0.001),并能显著降低活性氧水平和细胞凋亡率(P<0.05);③与单纯脂肪间充质干细胞相比,AMSC-Exos内miR-126-3p的mRNA相对表达量显著升高(P<0.001),与单纯人脐静脉内皮细胞相比,与脂肪间充质干细胞共培养可明显提高人脐静脉内皮细胞中miR-126-3p表达量(P<0.001);④miR-126-3p过表达可明显促进糖脂毒性人�BACKGROUND:MiR-126-3p from adipose-derived mesenchymal stem cells released exosomes(AMSC-Exos)has been used as a potential biomarker of diabetes,but few studies of glucolipotoxicity and mechanism of miR-126-3p on human umbilical vein endothelial cells are found.OBJECTIVE:To explore the effect and mechanism of miR-126-3p from AMSC-Exos on glucolipotoxicity of human umbilical vein endothelial cells.METHODS:(1)Glucolipotoxicity model of human umbilical vein endothelial cells was induced by 30 mmol/L glucose and 100μmol/L palmitic acid in vitro.(2)The supernatant of passage 4 AMSC-Exos was collected.The morphology of AMSC-Exos was observed by transmission electron microscope.The size distribution of AMSC-Exos was determined by nanoparticle tracking analysis.(3)Human umbilical vein endothelial cells with glucolipotoxicity were treated with AMSC-Exos.The content of reactive oxygen species and apoptosis rate were detected by flow cytometry.ELISA was implemented to detect the expression of inflammatory factors.(4)RT-qPCR was used to detect the expression levels of miR-126-3p mRNA in AMSC-Exos and normal human umbilical vein endothelial cells co-cultured with AMSC.(5)miR-126-3p overexpression was transfected into human umbilical vein endothelial cells.Cell proliferation was detected by CCK-8 assay.Western blot assay was used to detect the expression levels of apoptotic proteins.(6)The target genes of miR-126-3p were predicted and verified by bioinformatics software.Dual luciferase reporter gene assay was applied to verify and predict signaling pathway.(7)CRK overexpression was transfected into human umbilical vein endothelial cells with glucolipotoxicity.The expression levels of apoptotic protein and p-AKT were detected by western blot assay.(8)Human umbilical vein endothelial cell glucolipotoxicity model was co-treated with AKT pathway inhibitor RG7440 and miR-126-3p overexpression/AMSC-Exos.Apoptotic protein expression was detected by western blot assay and apoptosis rate was detected by flow cytometry.RESULTS AND CONC

关 键 词：脂肪间充质干细胞 外泌体 miR-126-3p 人脐静脉内皮细胞 糖脂毒性 AKT通路 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学]