【摘要】 目的:探究大鼠外周血间充质干细胞(PBMSC)的有效富集方法,并进一步研究其生物学特性。方法:采集经G-CSF动员的4周龄SD大鼠外周血,用密度梯度离心法分离出单个核细胞,继而采用贴壁培养法获得成纤维样集落生长细胞,绘制细胞生长曲线;应用流式细胞术(CD90、CD44、CD29、CD45、CD11b和CD79a)和免疫细胞化学染色法(CD73、CD105、CD34和HLA-DR)分析富集的第2代PBMSC的表型特征,并对其进行成骨、成软骨和成脂肪细胞诱导分化能力鉴定。结果:1原代培养6-7 d可见明显的成纤维样细胞集落生长,至培养21 d细胞生长汇合度可达80%;传代细胞贴壁能力强,形态均一,呈梭形漩涡状生长;传代培养的PBMSC于培养次日即可进入对数生长期,细胞倍增时间为39.2 h。2富集的PBMSC高表达CD90、CD44、CD29、CD73及CD105,不表达CD45、CD11b、CD79a、CD34和HLA-DR。3PBMSC分别经成骨、成软骨和成脂细胞诱导分化培养21 d后,再分别经茜素红染色显示有大量钙化结节形成,阿利新蓝染色的胞浆内有大量糖胺聚糖形成,油红O染色细胞内有大量脂滴形成。结论:采用本研究方法可从大鼠外周血中分离富集到高纯度、高丰度的PBMSC,且其具备MSC的生长特点和表型特征,并具有中胚层多系分化能力。更多还原

【Abstract】 Objective:To explore the effective method for enrichment of rat peripheral blood-derived mesenchymal stem cells(PBMSC) and study the cell biological characteristics.Methods:Peripheral mononuclear cells were isolated by density gradient centrifugation from blood of 4 week old rats after G-CSF mobilization.Thereafter,the fibroblast-like cells were acquired by plastic-adherent culture,and the proliferation curve was assayed.For analyzing surface markers of the second generation cultured isolated PBMSC,both flow cytometry(CD90,CD44,CD29,CD45,CD11 b and CD79a) and immunocy to chemical staining(CD73,CD105,CD34 and HLA-DR) methods were used.Furthermore,the differentiation capacities of PBMSC into osteocytes,chondrocytes and adipocytes were identified.Results:(1) The adherent cells displayed typical colony-forming unit fibroblast(CFU-F) growth pattern after 6-7 day of primary culture and reached 80%confluence after 21 days of culture.The passaged PBMSC possessed high proliferative capacity and spindle growth pattern and was able to grown into exponential phase next day with a doubling time of 39.2 h.(2)PBMSC expressed mesenchymal markers such as CD90,CD44,CD29,CD73 and CD105,but failed to expressed markers of CD45,CDllb,CD79 a,CD34 and HLA-DR.(3) After 21 days of culture in osteogenic,chondrogenic and adipogenic differentiation media,calcifying nodules,intracellular glycosaminoglycans and lipid droplets could be found by alizarin red,alcian blue and oil red-O staining,respectively.Conclusion:PBMSC can be enriched from rat peripheral blood with high purity and abundance by our methods.The growth and phenotypic characteristics of the isolated PBMSC are consistent with that of well-known MSC,and these cells possess the capability to multi-lineage mesoderm differentiation.更多还原