人胎盘间充质干细胞的体外分离纯化及成骨能力的研究

The purification and osteogenesis of human placenta-derived mesenchymal stem cells in vitro

目的通过体外分离纯化人足月胎盘间充质干细胞(hPMSCs),以研究其生物学特征及成骨能力。方法 采用胶原酶消化贴壁培养及人淋巴细胞分离液从人足月胎盘中分离纯化间充质干细胞(MSCs);流式细胞仪检测其细胞表面标志物的表达并运用MTT测定细胞生长曲线;电镜观察细胞超微结构;地塞米松、维生素C与β-甘油磷酸钠联合诱导其向成骨细胞分化,碱性磷酸酶及茜素红染色检测其碱性磷酸酶活性及钙结节。结果培养14天后可见大量贴壁细胞呈成纤维状生长,传代后增殖能力显著增强;强表达CD44,CD29,不表达CD34,CD45;经诱导后具有向成骨细胞分化的潜能,茜素红及碱性磷酸酶染色结果阳性。结论 人足月胎盘中同样含有MSCs,与其他来源的MSCs生物学特性相似,并且具有向中胚层细胞分化的能力,可作为组织工程及细胞替代疗法新的干细胞来源。

Objective To study the biological characteristics and osteogenic abilities of human placentaderived mesenchymal stem cells （hPMSCs） by isolation and purification of hPMSCs in vitro. Methods： hPMSCs were isolated and purified using collagenase digestion and adherent culture and human lymphocyte separation medium from full - month born human placenta. The expression of cell surface markers was detected using flow cytometry and cell growth curve was measured using MTT. Cell ultra-structure was observed using electron microscope, hPMSCs were induced to differentiate into osteoblasts using the combination of dexamethasone, vitamin C, and β-glycerophosphate. The alkaline phosphatase activity and calcium nodules were measured by alkaline phosphatase and Alizarin red staining. Results： A large number of adherent cells were visible as fibrous growth after 14 days culturing and exhibited strong proliferative ability after passage. Both CD44 and CD29 were expressed strongly in these cells, however, CD34 and CD45 were not expressed, hPMSCs were potent to differentiate into osteoblasts after induction, and the results of Alizarin red and alkaline phosphatase staining were positive. Conclusions ： MSCs from human fullmonth born placenta had simlilar biological characteristics of MSCs from other sources and had the potential to differentiate into mesoderm cells, hPMSCs could be used as a new source of stem cells in tissue engineering and cell replacement therapy.