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目的从胎盘组织中分离获得人胎盘源间充质干细胞(PMSCs),并诱导其向成骨细胞和脂肪细胞分化,为PMSCs应用于组织工程和细胞治疗提供实验依据。方法将人胎盘组织经胶原酶消化、贴壁和传代培养获得PMSCs,流式细胞仪检测其表面标志,分别联合应用β-甘油磷酸钠、维生素C、地塞米松体系和3-异丁基-1-甲基黄嘌呤、胰岛素、吲哚美辛、地塞米松体系将其诱导分化为成骨细胞和脂肪细胞,继而采用碱性磷酸酶检测和Von kossa染色对其进行成骨分化鉴定,采用油红O染色对其进行脂肪分化鉴定。结果从人胎盘中成功分离获得了PMSCs,形态呈纤维状,细胞增殖能力强;流式细胞仪分析显示PMSCs表达CD29、CD44、CD105、CD106和CD166,不表达CD34、CD45、HLA-DR;人PMSCs经成骨诱导2周后,碱性磷酸酶染色呈强阳性,Von kossa染色可见明显钙结节;人PMSCs经成脂诱导3周后,油红O染色呈阳性,有明显的脂滴出现。结论建立了有效分离、培养人PMSCs的实验体系,并能使其在体外诱导向成骨细胞和脂肪细胞分化,表明PMSCs可作为新型来源的种子细胞应用于组织工程。
OBJECTIVE: To isolate human placental derived mesenchymal stem cells (PMSCs) from placenta and to induce their differentiation into osteoblasts and adipocytes, and to provide experimental evidence for the application of PMSCs in tissue engineering and cell therapy. Methods Human placental tissue was digested by collagenase, adherent and subcultured to obtain PMSCs. Flow cytometry was used to detect the surface markers of them. Combined with β-glycerophosphate, vitamin C, dexamethasone and 3-isobutyl- 1-methylxanthine, insulin, indomethacin and dexamethasone were induced to differentiate into osteoblasts and adipocytes, then identified by alkaline phosphatase assay and Von kossa staining for osteogenic differentiation. Oil red O staining their fat differentiation identification. Results PMSCs were successfully isolated from human placenta and were characterized by fibrous morphology and strong cell proliferation. Flow cytometry analysis showed that PMSCs expressed CD29, CD44, CD105, CD106 and CD166 but not CD34, CD45 and HLA-DR After 2 weeks of osteogenic induction, PMSCs were strongly positive for alkaline phosphatase staining and obvious calcium nodules were found by Von kossa staining. After 3 weeks of adipogenic differentiation, PMSCs were positive for oil red O staining and obvious lipid droplets appeared . Conclusion The experimental system of effective separation and culture of human PMSCs was established and induced to differentiate into osteoblasts and adipocytes in vitro. It indicated that PMSCs could be used as new type of seed cells in tissue engineering.