人胎盘源间充质干细胞向成骨细胞和脂肪细胞分化的实验研究

Study on the Osteogenic and Adipogenic Differentiation from Human Placenta-derived Mesenchymal Stem Cells

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 placenta-derived mesenchymal stem cells（PMSCs） from placenta tissues and induce their osteogenic and adipogenic differentiation,which provides evidence for the application in tissue engineering and cell therapy.Methods The placenta tissues were di-gested by collagenase Ⅳ and cultured with low-glucose DMEM supplemented with b-FGF.The ad-herent cells were collected and passaged.The phenotypes of the cultured cells were detected by flow cytometry.The osteogenic differentiation was induced with β-glycerol phosphate disodium salt-pentahydrate,vitamin C,dexamethasone whereas the adipogenic differentiation was induced with 3-isobutyl-1-methylxanthine（IBMX）,insulin,indomethacin,dexamthasone.The osteogenic differentiation was detected with alkaline phosphate assay and von kossa staining.The adipogenic differentiation was observed with Oil red O staining.Results PMSCs were isolated and generated from human placenta successfully which were fibroblast-like cells with high proliferation.Flow cytometry analy-sis showed that PMSCs expressed CD29,CD44,CD105,CD106,CD166,and did not express CD34,CD45,HLA-DR.These cells were high positive for alkaline phosphate staining and also showed mineralization demonstrated with von kossa staining after two weeks culture induction of osteogenic differentiation.Futhermore,liquid vacuoles were detected by Oil red O staining after 3 weeks culture induction of adipogenic differentiation.Conclusion Effective culture system is es-tablished to isolate and generate PMSCs with the osteogenic and adipogenic differentiation,which indicated that PMSCs could be served as a new mesenchymal cell source in tissue engineering.