人脐带间充质干细胞外泌体miR-21-5p对颗粒细胞凋亡的影响  

Effects of miR-21-5p exosomes from human umbilical cord mesenchymal stem cells on apoptosis of granular cells

作　　者：杜生荣[1] 杨雷 孙艳[1] 林运鸿[1] 陈清汾 笪琳萃 黄鹏宇 郑备红[1] Du Shengrong;Yang Lei;Sun Yan;Lin Yunhong;Chen Qingfen;Da Lincui;Huang Pengyu;Zheng Beihong(Center of Reproductive Medicine,Fujian Maternity and Child Health Hospital,College of Clinical Medicine for Obstetrics&Gynecology and Pediatrics,Fujian Medical University,Fuzhou 350001,China;Obstetrics Department,Fujian Maternity and Child Health Hospital,Fujian Maternal-Fetal Clinical Medicine Research Center,Fuzhou 350001,China;Prenatal Diagnostic Center,Fujian Maternity and Child Health Hospital,Fujian Key Laboratory of Prenatal Diagnosis and Birth Defect Fujian Key Laboratory of Prenatal Diagnosis and Birth Defect,Fuzhou 350001,China)

机构地区：[1]福建省妇幼保健院生殖医学中心,福建医科大学妇儿临床医学院,福州350001 [2]福建省妇幼保健院产科,福建省母胎医学临床医学研究中心,福州350001 [3]福建省妇幼保健院产前诊断中心,福建省产前诊断与出生缺陷重点实验室,福州350001

出　　处：《中华生殖与避孕杂志》2024年第9期930-937,共8页Chinese Journal of Reproduction and Contraception

基　　金：福建省科技创新联合项目(2023Y9383);福建省自然科学基金(2021J01410);福建省科技重大专项项目(2021YZ034011);福建省卫生健康重大科研专项(2021ZD01002)。

摘　　要：目的:探究人脐带间充质干细胞来源的外泌体miR-21-5p对人颗粒细胞凋亡的影响。方法:不同浓度(0μmol/L、30μmol/L、60μmol/L和100μmol/L)磷酰胺氮芥作用KGN细胞48 h构建颗粒细胞凋亡模型,采用qPCR和Western blotting分别检测bax、bcl2的mRNA和蛋白水平,并用流式细胞计数检测凋亡率,以筛选出磷酰胺氮芥构建凋亡模型的最佳浓度。利用hsa-miR-21-5p过表达质粒瞬时转染人脐带间充质干细胞,通过qPCR检测hsa-miR-21-5p表达水平;分离过表达miR-21-5p的外泌体,利用纳米流式和电子显微镜鉴定miR-21-5p外泌体。对磷酰胺氮芥构建成功的KGN细胞凋亡模型中加入不同浓度(5μg/mL、10μg/mL和15μg/mL)的过表达miR-21外泌体,采用qPCR和Western blotting分别检测bax、bcl2的mRNA和蛋白水平,采用PKH-26标记外泌体示踪其在颗粒细胞的位置。结果:60μmol/L磷酰胺氮芥作用的KGN细胞中bax mRNA和蛋白水平均显著高于0μmol/L磷酰胺氮芥组(均P<0.001),bcl2 mRNA和蛋白水平均显著高于0μmol/L磷酰胺氮芥组(P=0.005,P<0.001)。60μmol/L磷酰胺氮芥作用后KGN细胞的凋亡率为(38.10±2.90)%,而30μmol/L磷酰胺氮芥干预后KGN细胞的凋亡率为(16.75±2.55)%,均显著高于0μmol/L磷酰胺氮芥组(P=0.020,P=0.006)。hsa-miR-21-5p瞬时转染人脐带血间充质干细胞,qPCR检测到has-miR-21-5p的表达高于未转染的空白对照组(P<0.001)。纳米流式检测外泌体表面蛋白CD9阳性率为14.9%,CD63阳性率为16.4%,CD81阳性率为31.4%;透射电子显微镜观察到外泌体呈“茶托型”;PKH-26标记的外泌体共聚焦显示进入颗粒细胞内并发挥生物学效应。5μg/mL、10μg/mL和15μg/mL空载质粒外泌体组与60μmol/L磷酰胺氮芥组比较bax mRNA表达水平差异均无统计学意义(均P>0.05)。但5μg/mL、10μg/mL和15μg/mL miR-21-5-p外泌体组bax mRNA表达水平低于60μmol/L磷酰胺氮芥组(P=0.008、P=0.003、P<0.001),而bcl2 mRNA表达在各组中差异均无统计学意�ObjectiveTo explore the effects of miR-21-5p exosomes derived from human umbilical cord mesenchymal stem cells on apoptosis of human granular cells.MethodsA granular cell apoptosis model was constructed by treating KGN cells with different concentrations(0µmol/L,30µmol/L,60µmol/L,and 100µmol/L)of phosphoramide nitrogen mustard for 48 h.The mRNA and protein levels of bax and bcl2 were detected using qPCR and Western blotting,respectively.The apoptosis rate was measured using flow cytometry to screen for the optimal concentration of phosphoramide nitrogen mustard for constructing an apoptosis model.Hsa-miR-21-5p overexpression plasmid was used for instantaneously transfecting human umbilical cord mesenchymal stem cells,and the expression level of hsa-miR-21-5p was detected by qPCR.The miR-21-5p exosomes were separated and identified by flow cytometry and electron microscope.Different concentrations(5µg/mL,10µg/mL and 15µg/mL)of miR-21 exosomes were added into successful KGN cell apoptosis model to overexpress miR-21.The mRNA and protein levels of bax and bcl2 were detected using qPCR and Western blotting,respectively.PKH-26 was used to trace the position of human granular cells.ResultsThe levels of bax mRNA and protein in KGN cells treated with 60µmol/L phosphoramide nitrogen mustard were significantly higher than those in the 0µmol/L phosphoramide nitrogen mustard group(all P<0.001),while the levels of bcl2 mRNA and protein were significantly higher than those in the 0µmol/L phosphoramide nitrogen mustard group(P=0.005,P<0.001).The apoptosis rate of KGN cells after 60µmol/L phosphoramide nitrogen mustard intervention was(38.10±2.90)%,while the apoptosis rate of KGN cells after 30µmol/L phosphoramide nitrogen mustard intervention was(16.75±2.55)%,they were all significantly higher than that of the 0µmol/L phosphoramide nitrogen mustard intervention group(P=0.020,P=0.006).Hsa-miR-21-5p was transiently transfected into human umbilical cord blood mesenchymal stem cells,and the expression of has-miR-21-5

关 键 词：人脐带间充质干细胞 外泌体 颗粒细胞 凋亡 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]