摘要
目的探讨人胚胎干细胞源性间充质干细胞(human embryonic stem cell derived mesenchymal stem cells,hESCMSCs)来源的外泌体的生物学特性。方法将已注册的未分化、人类ESC H9细胞系诱导成间充质干细胞(mesenchymal stem cells,MSCs),通过旋转超滤法从培养基中提取外泌体(exosomes)。流式细胞分析技术鉴定hESC-MSCs表面标志物,对间充质干细胞进行三系分化诱导,利用透射电镜、Izon qNano纳米粒子分析系统观察外泌体的形态和大小,Western blotting鉴定外泌体表面特异性标志物,观察hESC-MSCs-Exo对人微血管内皮细胞(human microvascular endothelial cells,HMEC-1)增殖和迁移的影响。结果成功诱导形成hESC-MSCs,并收集纯化外泌体。诱导后的MSCs CD29、CD73、CD90、CD105、CD146、CD44表达阳性,CD34、CD133、CD45、HLA-DR-PE表达阴性;hESC-MSCs通过诱导完成成骨、成脂和成软骨分化;hESCMSCs-Exo为粒径在40~100nm的囊泡,表达特异性的表面标志物CD9、CD63和CD81;体外促进HMEC-1的增殖和迁移。结论 hESC-MSCs-Exo作为一种生物活性囊泡,能够促进血管内皮细胞的增殖和迁移;hESC-MSCs-Exo来源无限,可为临床组织损伤的修复治疗提供丰富的干细胞外泌体来源。
Objective To investigate the biological characteristics of exosomes secreted by human embryonic stem cell-de- rived mesenchymal stem cells （hESC-MSCs-Exo）. Method The registered undifferentiated human ESC H9 cell line was induced into mesenchymal stem cells （MSCs） in a modified one-step method. Exosomes were collected by ultracentrifugation from the cul- ture medium of hESC-MSCs. The mesenchymal stem cells were identified by flow cytometry. The morphology and size of the exo- somes were observed by transmission electron microscopy （TEN/） and Izon qNano nanoparticles. Western blotting was used to iden- tify the exosome. The effects of hESC-MSCs-Exo on the proliferation and migration of human microvascular endothelial ceils （HMEC-1） were observed. Result hESC H9 cell line was efficiently induced into hESC-MSCs,and hESC-MSCs-Exo were col- lected and identified, hESC-MSCs were verified by positive markers for CD34, CD40, CD90, CD105, CD146, CD44 antigens, and negative markers for CD34, CD133, CD45 and HLA-DR-PE. And hESC-MSCs own the osteogenesis,chondrogenesis,and adipo- genesis multi-differentiation potential, hESC-MSCs-Exo is a vesicle with a diameter of 40-100nm,which expresses specific sur- face markers C139,CD63 and CD81 ,and promotes the proliferation and migration of HMEC-1 in Vitro. Conclusion hESC-MSCs- Exo, as a bioactive vesicle,can promote vascular endothelial cells proliferation and migration and has a wide range of sources for the treatment of tissue damage.
出处
《实验与检验医学》
CAS
2017年第2期143-147,共5页
Experimental and Laboratory Medicine
基金
国家自然科学基金资助项目
编号81471243
关键词
人胚胎干细胞源性间充质干细胞
外泌体
生物学特性
Human embryonic stem cell derived mesenchymal stem cells
Exosomes
Biological characteristics
