人脂肪间充质干细胞的原代培养及体外成骨成脂诱导分化

Osteogenic differentiation of primary cultured human adipose-derived mesenchymal stem cells accompanying adipogenic differentiation in vitro

目的:建立体外分离培养获得人脂肪间充质干细胞(human adipose-derived mesenchymal stem cells,hADSCs)的方法,并观察其形态、免疫表型、生物学特性。初步探讨其体外成骨过程中出现成脂现象的原因及机制。方法 :剖宫产手术获得腹部皮下脂肪,0.15%Ⅰ型胶原酶消化法获得人脂肪间充质干细胞并进行体外培养。行MTT细胞增殖实验绘制增殖曲线,使用流式细胞及细胞免疫荧光技术检测细胞表面抗原,行成骨成脂分化鉴定其分化潜能。通过RT-PCR技术检测成骨成脂过程中过氧化物酶体增殖物激活受体γ-2(peroxisome proliferator activated receptorγ-2,PPARγ-2)和骨桥蛋白mRNA表达情况。结果 :通过分离培养,获得了大量旋涡状生长的人脂肪间充质干细胞。MTT法显示细胞增殖能力强。流式细胞鉴定结果显示,CD29、CD73、CD105、CD166高表达,CD31、CD34、CD45、HLA-DR低表达。细胞免疫荧光结果与之相符。hADSCs在特定诱导条件下,具有成骨成脂分化潜能。在成骨分化过程中同时伴有成脂发生。RT-PCR结果显示,与对照组相比,成脂诱导组PPARγ-2 mRNA有时间依赖性递增表达,差异具有统计学意义(P<0.01)。与对照组相比,成骨诱导组骨桥蛋白,PPARγ-2 mRNA均有时间依赖性递增表达,差异具有统计学意义(P<0.01)。结论:建立了一种分离培养hADSCs简单可靠的方法。获得的细胞具有贴壁生长、增殖活性强、干细胞表型以及多向分化等特征。hADSCs体外成骨过程中,三酰甘油的形成对成骨具有一定的促进作用。

Objective： To establish the isolation and culture method of human adipose-derived mesenchymal stem cells（hADSCs） derived from human adipose in vitro,so as to explore their morphology,identify cell surface markers,observe biological properties,and discuss the possible causes and mechanism of the phenomenon of hADSCs′ osteogenic differentiation accompanying with synthesis of triglycerides.Methods： Human adipose tissue were obtained from abdominal operation.The hADSCs were isolated from human adipose tissue by 0.15% collagenase digesting.The cells were applied to do the experiments ： MTT method,flow cytometry,immunofluorescence.Its differentiation potential was proved by osteogenic and adipogenic differentiation.The osteogenic and adipogenic related genes： PPARγ-2,osteopontin expression were detected by real-time fluorescent quantitative PCR technique.Results： After isolation and culture,we obtained a large amount of hADSCs,which grew like swirls.MTT revealed high capability for and proliferation.The flow cytometry showed CD29＋,CD31-,CD34-,CD45-,CD73＋,CD105＋,CD166＋,HLA-DR-,which fit the results of immuno fluorescence.Moreover,these cells could be functionally induced into adipocytes and osteoblasts in the presence of appropriate conditioned media.During osteogenic differentiation,we found it accompanying with the synthesis of triglycerides.RT-PCR results proved that during the differention process,osteogenic and adipogenic related genes began to be expressed gradually,which had statistically significant（P＆lt;0.01）.Conclusion： Highly efficient isolation and cultivation methods for hADSCs have been developed.They are a kind of mesenchymal cells with great application prospect,which characterized with adherent growth,high proliferation,stem cell phenotype and multipotent differentiation.During vitro osteogenic differentiation,the triglyceride formation has a certain role in promoting osteogenesis.