诱导骨髓间充质干细胞向成骨细胞分化的实验研究

Induction of bone mesenchymal stem cells to form osteoblast

目的明确大鼠骨髓间充质干细胞(bone mensenchymal stem cells,BMSCs)在体外全骨髓培养条件下的生物学特性及其向成骨细胞分化的能力,探索更为简便有效的BMSCs体外培养方法。方法提取大鼠原代BMSCs,用αMEM完全培养基和成骨诱导条件培养基体外培养,倒置相差显微镜和HE染色观察细胞形态;台盼蓝活细胞计数绘制细胞生长曲线;ALP检测试剂盒检测ALP含量变化和von Kossa染色检测细胞矿化结节,以判断细胞是否向成骨细胞分化。结果全骨髓培养法在体外培养的大鼠原代BMSCs贴壁生长,呈梭形或多角形;使用Dex-SaMEM向成骨诱导后的BMSCs具有与成骨细胞在体外培养时相似的特征,倍增时间延长;合成ALP能力显著增强(P<0.05),von Kossa染色出现阳性的棕褐色或棕黑色团块的钙化结节。结论全骨髓培养法取材方便,经成骨诱导培养的BMSCs表现出成骨细胞的形态特征和生物学特性,该方法可作为骨组织工程种子细胞培养的常规方法。

Objective To identify the bone formation capability and biological characteristics of rat BMSCs and search for a simple and effective method to culture BMSCs in vitro. Methods BMSCs was cultured in vitro with SαMEM and bone formation conditioned medium,cell morphous was observed by inverted phase contrast microscope and HE stain; proliferation was detected by trypan blue viable count; bone formation was identified by changes of alkaline phosphatase contents in BMSCs and von Kossa. Results Rat BMSCs grew adherent, polygon when primary cultured by the method of whole bone marrow differential adherence. Meanwhile, When BMSCs was induced to osteoblast with dexamethasone, the same homoplastic characteristic was showed, the proliferative speed was stepped down,doubling generation time was prolonged,ALP content was enhanced obviously （P〈0.05） and calcified nodus were showed be positive by Von Kossa staining. Conclusion Whole bone marrow adherence screening method is a simple and effective method for isolating BMSCs and can be applied as a routine method to get seed cells for bone tissue engineering. BMSCs have a tendency to become osteoblast when induced.