诱导兔骨髓间充质干细胞定向分化为成骨细胞和软骨细胞的研究

Experimental studies of osteogenic and chondrogenic potentiality of rabbit bone marrow-derived mesenchymal stem cells

目的 :探讨兔骨髓间充质干细胞 (MSC)体外分离培养、增殖 ,以及向成骨细胞和软骨细胞定向诱导分化的条件 ,为骨、软骨缺损的修复和重建提供种子细胞。方法 :抽取兔髂骨及胫骨的骨髓液 ,经密度梯度离心法及塑料板贴壁培养法分离纯化出MSC ,并增殖。对第二代MSC进行成骨矿化诱导 ,测定其碱性磷酸酶 (ALP)活性及成骨矿化情况。并对第二代MSC进行诱软骨特定培养液诱导 ,对诱导细胞进行甲苯胺蓝染色 ,测定诱导后的软骨细胞表型。结果 :MSC为梭型的成纤维细胞样贴壁生长 ,增殖能力强。在成骨诱导剂作用下 ,MSC具有成骨能力 ,诱导后ALP活性明显提高 ,并且出现矿化结节沉积。在成软骨诱导剂作用下 ,可见细胞由成纤维样的梭形变为椭圆形及肾形 ,并分泌基质 ,甲苯胺蓝染色呈阳性 ,表现为软骨细胞分化特点。结论 :在特定诱导条件下 ,兔骨髓MSC体外可定向诱导分化为成骨细胞和软骨细胞 ,可用于组织工程骨和软骨组织的修复和重建。

Objective:To explore the optimal system of osteogenic and chondrogenic potential of rabbit bone marrow-derived mesenchymal stem cells(MSC).Methods:MSC were harvested from iliac and tibias crest and obtained by using gradient centrifuge and adhesive culture methods.l The passaged MSC were cutured in vitro under osteogenic and chondrogenic inductive environments,and their osteogenic and chondroˉgenic properties were investigated.Results:MSC in culture were uniformly spondle-shaped in appearance and showed active proliferative capacˉity.They could be induced to differentiate into osteoblasts under osteogenic induction.Alkaline phosphatase(ALP)activity of the osteogenic inˉductive cells significantly higher than that of control,and these cells could form mineralized nodules.Culture cells also could differentiate into chondrocytes after exposure to chondrogenic inductive medium,and the induced cells were positively stained by toluidine blue at various time points.Conclusion:The methods of isolating,culturing,and expanding rabbit MSC are feasible.The culture-expanded MSC have osteogenic and chondrogenic potentiality,so they are useful for bone and cartilage engineering by serving as optimal autogenous cell source.