碱性成纤维细胞生长因子对兔骨髓间充质干细胞向成骨细胞分化的影响

摘要

Objective To investigate the effects of basic fibroblast growth factor (bFGF) on differentiation of the rabbit's bone marrow mesenchymal stem cells (BMSCs) into osteoblasts,and to explore the optimal concentration of bFGF.Methods The BMSCs were cultured by the whole blood adherent method and then surface antibody (CD29,CD45 and CD45 by flow cytometry) and osteoblasts (by HE staining and alcian blue staining),and cartilage cells [by alizarin red staining and alkaline phosphatase (ALP) staining] were identified.The 2nd generated BMSCs were cultured with different concentrations (0,1,5,10,50,and 100 ng/mL) of bFGF.detecting alkaline phosphatase (ALP) with PNP colorimetric method and osteocalcin (OC) expression with radioimmunoassay (RIA) method on day 4,7,10 and 14 of culture.Results The positive rate of cell surface antigen CD29 was 92.23％,CD44 was 80.01％,and CD45 was 1.91％.HE staining showed that the cell morphology became polygonal or polygonal and there were multiple protrusions,the black particles could be seen within perinuclear cytoplasm.Alcian blue staining showed thata large number of azure materials diffused.A large number of red calcium nodules were seen by Alizarin red staining.The brown granules and massive precipitation in the cytoplasm were seen by ALP staining,which confirmed the cells were BMSCs with the potential of differentiating into cartilages and osteoblasts.The expression of ALP and OC in the 2nd generation of BMSCs was increased with different concentrations of bFGF as the time passes by.The expression of ALP and OC in BMSCs treated with 50 ng/mg bFGF was higher than that of BMSCs treated with 0,1,5,10 and 100 ng/mL bFGF at the same time (all P ＜0.01).Conclusion The bFGF promotes BMSCs differentiating into osteoblasts which is in a time-dependent manner and the optimal concentration is 50 ng/mg.%目的 观察碱性成纤维细胞生长因子(bFGF)对兔骨髓间充质干细胞(BMSCs)向成骨细胞分化的影响,并探讨其最佳作用浓度.方法 采用体外全血贴壁法培养兔BMSCs,进行表面抗原(CD29、CD44、CD45,采用流式细胞术)及成软骨(HE染色和阿尔新蓝染色)、成骨[茜素红染色及碱性磷酸酶(ALP)染色]分化潜能鉴定.取第2代BMSCs,加入含0、1、5、10、50、100 ng/mL bFGF的成骨诱导液,分别于培养第4、7、10、14天采用PNP比色法检测ALP表达,采用放射免疫分析法检测骨钙素(OC)表达.结果 细胞表面抗原CD29阳性表达率为92.23％,CD44阳性表达率为80.01％,CD45阳性表达率为1.91％;HE染色见细胞形态为多角形或多边形,有多个凸起,核周细胞质内可见黑色颗粒,阿尔新蓝染色可见细胞间大量天蓝色物质弥漫分布;茜素红染色可见大量红色钙结节,ALP染色可见细胞质中出现棕褐色的颗粒和块状沉淀;证实分离培养的细胞为BMSCs,并具有成软骨和成骨分化潜能.随着作用时间的延长,BMSCs经不同浓度bFGF作用后ALP及OC表达均呈升高趋势.相同作用时间下,BMSCs经50 ng/mL bFGF作用后ALP及OC表达均高于0、1、5、10、100 ng/mL bFGF作用后(P均＜0.01).结论 bFGF能促进BMSCs向成骨细胞分化,并呈时间依赖性;其最佳作用浓度为50 ng/mL.