全骨髓贴壁法分离培养rBMSCs及成骨诱导探讨

[摘要]　目的 通过研究在体外培养大鼠骨髓基质干细胞(rBMSCs)及其成骨生物学特性,探讨构建骨组织工程种子细胞的方法。方法 通过全骨髓贴壁培养法分离大鼠骨髓基质干细胞,应用含地塞米松、维生素C、β2甘油磷酸钠的诱导培养液定向诱导传代细胞向成骨细胞分化,并检测碱性磷酸酶活性及钙结节茜素红染色反应,进行成骨细胞鉴定。结果 全骨髓贴壁培养下,骨髓基质干细胞增殖能力强,生长迅速,呈成纤维细胞样生长。诱导条件下细胞的碱性磷酸酶活性明显增高,并且钙结节茜素红染色反应阳性。结论 采用全骨髓贴壁培养法,骨髓基质干细胞分离培养及体外扩增更为简便迅速,诱导条件下成骨能力肯定,可为骨组织工程构建提供大量种子细胞。

Induction of osteogenesis in rat Bone marrow mesenchyma stem cells by whole bone marrow adherent culture

Abstract:Objectives: To observe the osteogenic ability and biological characteristics of rat bone mesenchymal stem cells (rBMSCs) in vitro and identify an approach to culturing seed cells that maybe better for bone tissue engineering construction. Methods: rBMSCs were isolated and purified by whole bone marrow wall adhesion culture procedure and passaged in vitro, which will be induced into osteoblastic cells using culture medium including dexamethasone, ascorbic acid and β-glycerophosphate.The results of osteogenesis were examined by Alkaline Phosphatase (ALP) staining reaction and calcium nodus alizarin red staining reaction. ALP enzyme activity of the cell layer was also measured. Results: In the conditions of whole bone marrow wall adhesion culture procedure, rBMSCs developed into fibroblast-like cells, growed rapidly and showed strong proliferation ability . The adherent cultured cells increased the activity of ALP, ALP staining reaction and calcium nodus alizarin red staining reaction were positive with the osteogenic inducer.Conclusions:Using the whole bone marrow wall adhesion culture procedure, the isolation and amplification of MSCs become more easily and rapidly in vitro. The Osteogenic Potential of cultured MSCs is stable with the osteogenic inducer. It could provide ideal seed cells for bone tissue engineering construction.