青少年特发性脊柱侧凸骨髓间充质干细胞分离培养及体外诱导分化为脂肪细胞

Isolation and culture of human bone marrow mesenchymal stem cells from adolescent idiopathic scoliosis patients and adipocyte induction in vitro

目的建立青少年特发性脊柱侧凸(adolescent idiopathic scoliosis,AIS)来源的骨髓间充质干细胞(mesenchymal stem cells,MSCs)分离、培养、传代的方法,并观察其体外成脂诱导潜能,为后续的AIS来源MSCs诱导分化过程中基因表达差异的比较研究作准备。方法采用密度梯度离心法分离出MSCs后培养并传代,利用流式细胞仪检测MSCs细胞表面标记物,用含1μmol/L地塞米松、10 mg/L重组人胰岛素、0.5 mmol/L 1-甲基-3-异丁基-黄嘌呤、100μmol/L吲哚美辛的诱导培养基对传至第3代的MSCs进行成脂诱导,诱导至第14天行油红O染色鉴定。结果 AIS来源骨髓标本分离培养的细胞经流式细胞术鉴定均显示CD29、CD44、CD105为阳性,CD34、CD45为阴性,符合MSCs的表型特征;MSCs经成脂诱导后均可分化为脂肪细胞,油红O染色阳性,可见胞质内呈橘红色的大小不一脂滴。结论密度梯度离心法可成功分离出高纯度的AIS来源的MSCs,其在体外具有良好的成脂潜能。本研究结果可为后续的MSCs诱导分化过程中基因表达差异研究打下基础。

Objective To establish an experimental isolation and culture of human bone - derived mesenchymal stem cells （hBMSCs） from patients with adolescent idiopathic scoliosis （AIS）, and to investigate their adipogenic diffe- rentiation potential in vitro. Methods The hBMSCs were isolated and cultured by the density gradient centrifugafion method, and subsequently identified by the flow eytometry method. The third passage hBMSCs were induced by adipogenie supplements （ 1 μmol/L dexamethasone, 10 mg/L insulin, 0. 5 mmol/L isobutylmethylxanthine, and 100 μmol/L indo- methacin）. Oil red - O solution was used for detection of lipid droplets in the induced cells. Results All of the hBMSCs with positive CD29, CD44, CD105 and negative CD34, CD45 were detected by the flow cytometry, which were in accor- dance with the characteristics of MSCs. Red - stained lipid droplets were observed in adipogenie differentially cultured hBMSCs from AIS with oil red - O staining. Conclusion Highly purified hBMSCs is successfully obtained with density gradient centrifugation method from AIS patients, with adipogenic potential in vitro. This paves the way for further studies of AIS.