大鼠外周血间充质干细胞的富集及生物学特性研究

Enrichment and Biological Characteristics of Peripheral Bloodderived Mesenchymal Stem Cells in Rats

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。

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, CIM5, CDllb and CD79a） and immunocytochemical 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 CDg0, CD44, CD29, CD73 and CD105, but failed to expressed markers of CD45, CD1 lb, CD79a, CD34 and HLA-DR. （3） After 21 days of culture in osteogeuic, 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.