凝血酶活化的富血小板血浆促进人骨髓间充质干细胞增殖

Thrombin-Activated Platelet-Rich Plasma Promotes the Proliferation of Human Marrow Mesenchymal Stem Cells

目的探索凝血酶活化的富血小板血浆(Thrombin-activated platelet-rich plasma,tPRP)替代牛血清,进行人骨髓间充质干细胞(Mesenchymal stme cells,MSC)分离及培养扩增的可行性。方法分别用MTT法和羧基荧光素二醋酸盐琥珀酰亚胺酯标记细胞技术,观察人骨髓MSC在含有tPRP和胎牛血清培养体系中的增殖状态:利用流式细胞学技术检测细胞表面表型;细胞化学染色法分析不同培养体系所获细胞的成骨及成脂细胞分化能力。结果tPRP培养的人骨髓MSC呈典型的成纤维细胞样形态,且tPRP促MSC增殖能力优于筛选后的胎牛血清。tPRP培养的MSC均表达CD29、CD73、CD166和HLA-ABC,而不表达CD31、CD34、CD45和HLA-DR。体外分化实验显示,利用tPRP培养的MSC具有体外成骨和成脂能力。结论tPRP可以替代胎牛血清,用于人骨髓MSC的培养。

Objective To investigate whether the supernatants of thrombin-activated platelet-rich plasma could be used as a substitute for the isolation and culture-expansion of human mesenchymal stem cells. Methods Human marrow mesenchymal stem cells （MSC） were cultured in media containing the supernatants of thrombin-activated platelet-rich plasma （tPRP） and fecal calf sera （FCS） selected from lots. Their proliferation status was evaluated by MTT and carboxyfluorescence diacetatesuccinimidyl ester-labeling assays. Furthermore, the phenotypic characteristics were analyzed by flow cytometry and the differentiations along adipogenic and osteogenic pathways were assessed by histological staining. Results MSC cultured in medium containing tPRP displayed a typical fibroblast-like morphology and, compared with （FCS）, tPRP exhibited more potent activities to support the expansion of MSC. Flow cytometry showed that they were homogenously positive for CD29, CD73, CD166 and HLA-ABC and negative for CD31, CD34, CD45 and HLA-DR. Differentiation assays showed that of MSC cultured with tPRP occupied the capacities of in vitro osteogenesis and adipogenesis. Conclusion It is concluded that human tPRP might be an optional substitute to FCS for MSC proliferation.