大鼠血管内皮祖细胞培养及鉴定  

Isolation and Culture of Endothelial Progenitor Cells Derived from Bone Marrow in Rats

作　　者：朱江[1] 刘煜敏[1] 孔朝红[1] 道文欣[1] 

机构地区：[1]武汉大学中南医院

出　　处：《中西医结合心脑血管病杂志》2010年第5期563-565,共3页Chinese Journal of Integrative Medicine on Cardio-Cerebrovascular Disease

基　　金：国家自然科学基金资助项目(No.30470601)

摘　　要：目的探索骨髓来源的血管内皮祖细胞的分离培养方法,为缺血性疾病治疗找到新的移植细胞来源。方法采集SD雄性大鼠骨髓细胞,用梯度密度离心法分离单核个细胞,种植于提前包埋了纤维连接蛋白的培养皿中培养,将血管内皮生长因子(VEGF)、重组纤维细胞生长因子(bFGF)和重组上皮生长因子(EGF)作为诱导剂,将72h后贴壁细胞(A组)和非贴壁细胞(B组)分别培养,观察细胞形态的变化以及数量变化,取A组在第14天,应用免疫组化和免疫荧光技术鉴定内皮祖细胞系列标志。结果A组细胞经过体外诱导后大量扩增,在28d时仍有强大扩增的活力,呈铺路石样,而B组细胞短期内可以大量增殖,呈长索样,之后逐渐变少;在细胞迁移实验中,A组细胞可以相互融合形成血管结构,而B组细胞之间则不能融合,随着时间推移,逐渐凋亡;A组经过CD34免疫荧光、CD133、FLK-1免疫组化鉴定呈阳性,并能特异性吸附FITC-UEA-和内吞DIL-Ac-LDL。结论自体骨髓细胞通过贴壁换液后可以分离培养出内皮祖细胞,取贴壁细胞经过体外诱导后大量扩增,并通过表面标记物鉴定可以确认为内皮祖细胞,而非贴壁细胞不能大量增殖,故将贴壁分离方法来筛选内皮祖细胞,此方法简易,并能满足细胞移植的需要,因而,在移植治疗脑缺血引起的内皮损伤应该有较好的临床应用前景。Objective To explore the procedures of isolation and cultivation of endothelial progenitor cells（EPCs）derived from bone marrow in rats.Methods EPCs were isolated from male Sprague-Dawley rat by Ficoll density gradient centrifugation.Discs were coated with fibronectin before EPCs were cultured in M199 in vitro,which induced by vascular endothelial growth factor（VEGF）,basic fibroblast growth factor（bFGF）and epidermal growth factor（EGF）.After 72 hours,adherent cells（A group）and nonadherent cells（B group）were cultured respectively.The morphous features of cells were studied.The expression of CD34＋,CD133＋ and Flk-1＋ after 14 days were detected by immunocytochemistry and immunofluorescence assays.Results Cultured adherent cells（A group）still possessed robust proliferative potential at 28 days,they had proliferative activity and appeared cobble-stone shape,which were formed vascular structure,and stained positively for CD133,Flk-1.They were expressed CD34 partly.The experiment of FITC labeled UEA adsorption and DiL-acLDL internalization was positive by fluorescence confocal microscopy.Whereas nonadherent cells（B group）grow rapidly at short period and gradually reduced.At 28 days,most cells were apoptosis and vascular structures could not form.Conclusion EPCs could be isolated from bone marrow though attachment-changing culture methods.Adherent cells possessed robust proliferative potential,and their number could reach the seeding cells criteria that were required in implantation in vivo.They could be used as an auto-donated seeding cell resource for the treatment of vascular dysfunction after stroke.

关 键 词：内皮祖细胞 骨髓 细胞培养 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]