诱导人骨髓间充质干细胞向成骨细胞和内皮细胞分化

Induction of differentiation of human mesenchymal stem cells into osteoblasts and endothelial cells

目的探索体外诱导人骨髓间充质干细胞(hMSCs)向成骨细胞和内皮细胞分化形成成骨细胞及内皮细胞共存体系的潜能和条件。方法采用密度梯度离心法分离培养hMSCs,采用含30 mg/L内皮细胞生长添加剂的内皮细胞诱导液诱导1周,然后更换为含1×10-8mol/L地塞米松、10 mmol/Lβ-甘油磷酸钠、30μg/mL维生素C的成骨细胞诱导液继续诱导7～14 d。诱导前以只加10%FBS的HDMEM培养基作为对照。采用流式细胞术检测细胞表面分子,通过倒置显微镜观察细胞形态学变化。向成骨细胞诱导14 d后,采用茜素红染色观察钙结节形成。结果经流式细胞术检测,诱导前分离培养的hMSCs表达CD90、CD105和CD44,不表达内皮细胞表面标志CD34和CD133,也不表达HLA-DR。诱导14 d后,hMSCs表面标志CD90和CD105的表达下降(P<0.05),成骨细胞表面标志CD44和HLA-DR及内皮细胞表面标志CD34和CD133增加(P<0.05)。诱导过程中原先长梭形的细胞缩短,细胞出现分层。hMSCs向成骨细胞诱导14 d后,茜素红染色显示有钙结节形成。结论hMSCs先后经内皮细胞诱导液和成骨细胞诱导液诱导,可在体外向内皮细胞和成骨细胞定向分化,形成内皮细胞和成骨细胞共存体系。

Objective To explore the potentials and conditions for the differentiation of human mesenchymal stem cells （hMSCs） into osteoblasts and endothelial cells in vitro. Methods hMSCs were isolated and cultivated by density gradient centrifugation method. The cells were treated with 30 mg/L endothelial cell growth supplement for one week, and were continued to be induced with 1 × 10^-8 mol/L dexamethasone, 10 mmol/L 13-sodium glycerophosphate and 30 ug/mL vitamine C for 7 to 14 days. HDMEM culture medium with 10% FBS before induction was served as control. The expression of cell surface antigens was detected by flow cytometry, and the morphological changes were observed by inverted microscopy. The cells were stained with alizarin red for the observation of calcium nodule. Results It was revealed by flow cytometry that the isolated and cultivated hMSCs expressed CD90, CD105 and CD44, and did not express CD34, CD133 and HLA-DR before induction. Fourteen days after induction, the expression of CD90 and CD105 decreased （ P 〈 0.05） , and that of CD44, HLA-DR, CD34 and CD133 increased （P 〈 0. 05）. During induction, the spindle-shaped cells contracted, and overlay was observed. Fourteen days after induction, calcium nodule formation was demonstrated by alizarin red staining. Conclusion hMSCs consecutively treated with endothelial cell inductor and osteoblast inductor can differentiate into endothelial cells and ostelblasts in vitro, forming the co-culture system of endothelial cells and osteoblasts.