人蜕膜间充质干细胞来源外泌体对高糖老化人真皮成纤维细胞功能的影响及其可能机制  

Effect of human decidua mesenchymal stem cells-derived exosomes on the function of high glucose-induced senescent human dermal fibroblasts and its possible mechanism

作　　者：苏建隆 马奎[2] 张翠萍 付小兵 Su Jianlong;Ma Kui;Zhang Cuiping;Fu Xiaobing(School of Medicine,Nankai University,Tianjin 300071,China;Research Center for Wound Repair and Tissue Regeneration,Medical Innovation Research Department,the PLA General Hospital,Beijing 100853,China;Research Unit of Trauma Care,Tissue Repair and Regeneration,Chinese Academy of Medical Sciences,2019RU051,Beijing 100048,China)

机构地区：[1]南开大学医学院,天津300071 [2]解放军总医院医学创新研究部创伤修复与组织再生研究中心,北京100853 [3]中国医学科学院严重创伤救治和组织修复与再生医学创新单元2019RU051,北京100048

出　　处：《中华烧伤与创面修复杂志》2022年第2期170-183,共14页Chinese Journal of Burns And Wounds

基　　金：北京市自然科学基金(7202197);军队医学科技青年实训计划(21QNPY128)。

摘　　要：目的建立人真皮成纤维细胞(HDF)高糖老化模型,探讨人蜕膜间充质干细胞(dMSC)来源外泌体对高糖老化HDF增殖、迁移、凋亡的影响及其可能机制。方法采用实验研究方法。收集2021年1—3月解放军总医院第四医学中心收治的4例男性包茎患者(18~22岁)环切术后废弃包皮组织,分离培养获取原代HDF。取第6代HDF,按照随机数字表法分为低糖组和高糖组,分别采用低糖完全培养基和高糖完全培养基进行每72小时换液、不传代培养,10 d后取细胞,于接种后24 h,采用β-半乳糖苷酶试剂盒检测细胞衰老情况;于接种后48 h,采用蛋白质印迹法检测细胞衰老相关蛋白p16、p53表达情况;于接种后24、48、72 h,采用细胞计数试剂盒8(CCK-8)法检测细胞增殖情况;于接种后48 h,采用脱氧尿嘧啶核苷(EdU)染色法检测细胞增殖情况,采用流式细胞术检测细胞周期及凋亡情况;于接种后24 h,采用Transwell实验测定细胞迁移能力。取人dMSC培养48~72 h,采用差速高速离心法获取其外泌体,采用透射电子显微镜观察dMSC外泌体形态,采用纳米颗粒追踪分析法检测dMSC外泌体的粒径分布,采用蛋白质印迹法检测dMSC外泌体标志蛋白CD9、肿瘤易感基因101(TSG101)的表达。取dMSC外泌体及前述高糖完全培养基诱导老化的HDF共孵育24 h,采用PKH67试剂盒检测细胞摄取外泌体的情况。取前述高糖完全培养基诱导老化的HDF,同前分为单纯高糖组、高糖+低浓度外泌体组、高糖+高浓度外泌体组,分别于高糖完全培养基中加入等体积的磷酸盐缓冲液、终质量浓度为50μg/mL dMSC外泌体、终质量浓度为100μg/mL dMSC外泌体进行常规细胞培养。分组后同前于对应时间点采用CCK-8法和EdU染色法、流式细胞术、Transwell实验分别检测细胞增殖、细胞周期和凋亡及细胞迁移情况。根据前述结果,另取经高糖完全培养基诱导老化的HDF,分为单纯高糖组、高糖+高浓Objective To establish a high glucose senescent model of human dermal fibroblasts(HDFs),and to investigate the effects of exosomes derived from human decidua mesenchymal stem cells(dMSCs)on the proliferation,migration,and apoptosis of senescent HDFs and possible mechanism.Methods The experimental research method was used.From January to March 2021,discarded foreskin tissue was collected for isolation and culture of primary HDFs from 4 male phimosis patients(aged 18-22 years)admitted for circumcision in the Fourth Medical Center of the PLA General Hospital.The 6th passage of HDFs were taken and divided into low glucose group and high glucose group according to the random number table,and subsequently cultured in low-glucose complete medium and high-glucose complete medium,respectively,with medium changed every 72 h without subculturing.After 10 days of culture,the cells were taken and measured for cellular senescence using theβ-galactosidase kit at 24 h after seeding;the expression of senescence-related proteins p16 and p53 was assessed by Western blotting at 48 h after seeding;cell proliferation was detected at 24,48,and 72 h after seeding using the cell counting kit 8(CCK-8)method;the cell proliferation was evaluated by 5-ethynyl-2'-deoxyuridine(EdU)staining method,cell cycle and apoptosis were measured by flow cytometry after 48 h of seeding;Transwell experiment was used for the calculation of cell migration rate at 24 h after seeding.The human dMSCs were taken and cultured for 48-72 h from which the exosomes were extracted by differential high speed centrifugal method.The morphology of dMSC exosomes was observed by transmission electron microscopy,the particle size distribution of dMSC exosomes was measured by nanoparticle tracking analysis,and the expression of dMSC-exosomes marker proteins CD9 and tumor susceptibility gene101(TSG101)were detected by Western blotting.The dMSC exosomes and high-glucose complete medium-induced senescent HDFs were co-cultured for 24 hours,then PKH67 kit was used to detect the u

关 键 词：间质干细胞 外泌体 细胞衰老 葡萄糖溶液 高渗 成纤维细胞 

分 类 号：R641[医药卫生—外科学]