组织工程与重建外科杂志

组织工程与重建外科杂志 ›› 2016, Vol. 12 ›› Issue (5): 269-280.doi: 10.3969/j.issn.1673-0364.a447

• 论著 •    下一篇

骨髓间充质干细胞与内皮细胞平面共培养促进微血管网形成的初步研究

张磊，付炜，张文，白洁，冯蓓，唐梓清，张海波   

  1. 上海交通大学医学院附属上海儿童医学中心心胸外科；上海交通大学医学院附属上海儿童医学中心儿科转化医学研究所
  • 发布日期:2020-07-23

Two-D imensional Co-Culture of Bone M arrow Mesenchymal Stem Cells and Endothelial Cells for Inducing the Self-Assemb led Vascular Network

ZHANG Lei1,FU Wei1,2,ZHANG Wen1,BAI Jie1,2,FENG Bei1,2,TANG Ziqing1, ZHANG Haibo1.   

  1. 1 Department of Pediatric Cardiothoracic Surgery;2 Institute of Pediatric Translational Medicine,Shanghai Children's Medical Center,School of Medicine,Shanghai Jiaotong University,Shanghai200127,China.Corresponding author: ZHANGHaibo E-mail:haibo.z@yahoo.com).
  • Published:2020-07-23
  • Contact: 国家自然科学基金（31200735）；上海市科委基金（134119a0400，15411966800）；上海卫生和计划生育委员会项目（20144Y0166）；东南大学生物电子学国家重点实验室开放研究基金。

摘要： 目的 研究大鼠骨髓间充质干细胞(BMSCs)与内皮细胞(ECs)在不使用Matrigel等基质,以及在含或不含血清的条件下,于普通平面培养皿上共培养的成网能力,为日后两类细胞相互作用的研究提供一种外界干扰最少的共培养条件。方法 以全骨髓贴壁培养法分离培养大鼠BMSCs,并从培养形态、细胞表面标记物和三系分化能力等方面予以鉴定。以添加或不添加10%FBS的低糖DMEM为培养液,将大鼠BMSCs与ECs共培养于普通平面培养皿上,连续观察微血管网的形成过程,并通过CM-Dil标记特定细胞的方法了解血管网中细胞构成。平滑肌细胞标记物SM22α,Calponin及内皮细胞标记物CD31被用于检测上述条件下微血管网中细胞标记物变化。结果 通过全骨髓贴壁培养可分离并获得大鼠BMSCs。在含10%FBS条件下,细胞于普通平面培养皿上共培养8 d可形成明显的微血管网,微血管网由大鼠BMSCs和ECs共同构成;在无血清共培养条件下,大鼠BMSCs和ECs可于24 h内共同参与形成微血管网。免疫荧光染色显示CD31+细胞和SM22α+/Calponin+细胞共同构成网状结构。结论 在普通平面培养皿上,以含血清或不含血清的培养液共培养大鼠BMSCs和ECs均可形成微血管网。

关键词: 骨髓间充质干细胞, 内皮细胞, 共培养, 微血管网

Abstract: Objective To observe the vascular network formation by co-culturing rat bone marrow mesenchymal stem cells(BMSCs)and endothelial cells(ECs)under conditions of two-dimensional co-culturing with/without serum,which would provide a minimum basal co-culture condition for the further study of the interactions between the cells.M ethods Rat BMSCs were isolated by direct adherence of the whole bonemarrow,and were identified by cellmorphology,cell surface antigens and three-lineage differentiation.Low-glucose DMEM with/without 10%FBSwas used during the co-culture of rat BMSCs and ECs,followed by a consecutive observation of the vascular network formation.CM-Dil solely labeled rat BMSCs or ECs were then co-cultured to verify the cell type within the network.Finally,immunofluorescent staining of EC marker CD31 and smoothmuscle cellmarker SM22αand Calponin was used to identify the cell differentiation during the co-culture. Resu lts Whole marrow direct adherence was used to isolate rat BMSCs successfully.With medium containing 10%FBS, co-cultured cells assembled vascular network in 8 days,which was formed together by rat BMSCs and ECs.In serum-free medium,however,self-assembled vascular network was formed together by co-cultured cells within 24 hours.In both settings,CD31+cells and SM22αor Calponin weremostly seen in the network structure.Conclusion Rat BMSCs and ECscan be co-cultured on two-dimensional plateswith/without serum-containingmedium for the induction of the self-assembled vascular network.

Key words: Bonemarrow mesenchymal stem cell, Endothelial cell, Co-culture, Vascular network

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