摘要
背景:目前骨髓间充质干细胞已经应用于多种组织血管的修复,但涉及尿道血管组织的报道甚少。目的:探讨体外诱导大鼠骨髓间充质干细胞分化为血管内皮细胞的可行性。设计、时间及地点:细胞学体外观察,于2007-07/2008-01在苏州大学附属儿童医院儿科研究所完成。材料:清洁级4周龄SD大鼠4只,由苏州大学实验动物中心提供。作为诱导剂的血管内皮细胞生长因子、碱性成纤维细胞因子为Sigma公司产品。方法:密度梯度离心法分离培养大鼠骨髓间充质干细胞,待细胞达80%～90%融合时消化传代。传至第3代,细胞按1×109L-1密度接种,加入含血管内皮细胞生长因子、碱性成纤维细胞因子的DMEM培养基向血管内皮细胞诱导分化。主要观察指标:MTT测定细胞生长曲线,免疫荧光法检测细胞表面CD44与Vimentin的表达,光学倒置显微镜下观察诱导后细胞形态变化,电镜观察细胞超微结构。结果:骨髓间充质干细胞生长曲线呈倒"S"型,第3代细胞表面CD44和Vimentin表达阳性率分别为89.3%和85.6%。向血管内皮细胞诱导1～3d细胞呈梭形或纺锤形,7d后变成不规则的圆形或椭圆形,胞浆淡染,胞核较大,核染色质粗,且部分细胞死亡,14d后整瓶细胞呈漩涡状生长,21～25d后细胞密集处呈内皮细胞特有的铺路石样排列。诱导后第21天,细胞浆内可见微管、微丝及W-P小体。结论:血管内皮细胞生长因子与碱性成纤维细胞因子联合诱导条件下,骨髓间充质干细胞在体外可分化为血管内皮细胞。
BACKGROUND： At present, bone marrow mesenchymal stem cells （BMSCs） have bean applied to repairing tissue vessels, but repairing urethral is less. OBJECTIVE： To investigate the feasibility of in vitro differentiation of rat BMSCs into vascular endothelial cells. DESIGN, TIME AND SETTING： The cytology in vitro study was conducted at the Institute of Pediatrics, Children＇s Hospital, Soochow University from July 2007 to January 2008. MATERIALS： A total of 4 clean Sprague Dawley rats aged 4 weeks were supplied by Animal Experimental Center, Soochow University. Vascular endothelial cell growth factor and basic fibroblast growth factor （Sigma, USA） were used in this study. METHODS： Rat BMSCs were isolated and cultured by the density gradient centdfugation, digested after 80%-90% of cells were confluent. At passage 3, cells at 1×10^9/L were incubated in DMEM, supplemented with vascular endothelial cell growth factor and basic fibroblast growth factor, and induced to differentiate into vascular endothelial cells. MAIN OUTCOME MEASURES： Growth curve was detected by the methyl thiazolyl tetrazolium （MTT） assay. CD44 and Virnentin expression on the cell surfaces was detected using immunofluorescence method. Cell morphology following induction was observed with an optical inverted microscope. Cell ultrastructure was observed under an electron microscope. RESULTS： Growth curve of BMSCs showed ＂S＂ shape. At the third passage, BMSCs were positive for CD44 （89.3%） and Vimentin （85.6%）. The induced cells became spindle shape after 1 to 3 days, which changed into irregular round or ellipse after 7 days, which contained light endochylema, lager nucleus and thickness nuclear chromatin. Some induced cells were dead. The induced cells grew in whirlpool shape after 14 days. BMSCs changed from spindle like cells to cobbles tone-like cells after 21 to 25 days. At day 21, induced cells could find microtubules and microfilament in endochylema, in which the Weibel-palade body was observed. CONCLUSION： BMSCs can be induced into vascular endothelial cells in vitro under the combined induction of vascular endothelial cell growth factor and basic fibroblast growth factor.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第19期3698-3702,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
苏州市社会发展科技计划项目(SS08021)
第五批苏州大学医学发展基金项目~~
