不同代次大鼠骨髓间充质干细胞和脂肪间充质干细胞成骨分化比较

Comparison on osteogenic differentiation ability of different passages of the mesenchymal stem cells derived from adipose and bone marrow in rats

本研究旨在观察不同代次骨髓间充质干细胞(BMSCs)和脂肪间充质干细胞(ADSCs)体外培养的生长特点和体外诱导成骨能力。通过密度梯度离心和贴壁培养法分离培养大鼠骨髓间充质干细胞和脂肪间充质干细胞,用含地塞米松、抗坏血酸、β-甘油磷酸钠的培养液定向诱导传代细胞向成骨细胞分化,并利用茜素红染色、碱性磷酸酶染色及PCR方法检测成骨细胞。结果表明骨髓及脂肪间充质干细胞呈成纤维细胞样生长,增殖能力强,生长迅速。第5、10、15、20代BMSCs及ADSCs经诱导培养后茜素红染色呈阳性并且出现"矿化"、碱性磷酸酶活性强,随着细胞代次的递增,诱导后细胞碱性磷酸酶活性呈递减趋势;诱导后的两类细胞传代后细胞仍能继续分化,并形成正常的"矿化"结节,且碱性磷酸酶染色均弱于初次诱导。结果提示,BMSCs及ADSCs易于分离培养及体外扩增,诱导条件下成骨能力强且成骨细胞传代培养仍具有成骨能力,适合作为再生医学骨组织工程的种子细胞。

The aim of study is to investigate the culture characterizations and osteoblast differentiation in vitro of the mesenchymal stem cells derived from adipose and bone marrow. The bone marrow stromal stem cells （BMSCs） and adipose derived stromal cells （ADSCs） were isolated using density gradient centrifuge method and cultured in vitro, followed by osteogenic cultivation in DMEM supplemented with dexamethasone, ascorbic acid and β-glycerophosphate. The osteogenic differentiation was assessed with alkaline phosphatase activity （ALP） alizarin red staining. The results showed that the morphology of BMSCs and ADSCs showed fibroblast-1 ike characterizations. The BMSCs and ADSCs of the 5, 10, 15 and 20 generations showed the positive results of ALP and Alizarin Red. At the same time, with passages raised; ALP activity decreased. Mineralized nodules of osteoblasts in BMSCs of 20 generations were more than those in other passages, but there was no difference in ADSCs. It suggest that the mesenchymal stem cells derived from adipose and bone marrow from rat can differentiate to osteoblasts when cultured in vitro and can be served as optimal autogenous cell source for bone tissue engineering.