摘要
目的观察碱性成纤维生长因子（bFGF）、表皮生长因子（EGF）和血小板衍生因子（PDGF－BB）对小鼠骨髓间充质干细胞（BMSCs）体外增殖和成骨分化的影响。方法采用贴壁培养纯化小鼠BMSCs，培养液（DMEM／F12，10％胎牛血清，青、链霉素）中添加（实验组）或者不添加（对照组）细胞因子bFGF、EGF和PDGF－BB（浓度均为10μg/L）进行培养，取传至第3代的细胞进行成骨分化，成骨诱导液：1×10-8mol／L地塞米松、50μmol／L抗坏血酸、10mmol／Lβ-甘油磷酸钠。流式细胞仪检测细胞表面标记抗原CIM5和CD90的表达；SYBgreen荧光定量聚合酶链反应（PCR）扩增检测成骨细胞特异性基因骨钙素和I型胶原的表达；碱性磷酸酶染色和茜素红染色检测成骨细胞的分化。结果初始接种后，对照组的小鼠BMSCs贴壁较快，而传代后实验组的小鼠BMSCs增殖较快。对照组和实验组的小鼠BMSCs经传代纯化后CIM5、CD90的阳性表达率分别为（27．344-0．69）％、（41．93±0．34）％和（0．48±0．12）％、（82．78±0．25）％，实验组的小鼠BMSCs在传代过程中较少观察到细胞的老化现象，细胞的纯化程度较高，而且其向成骨分化的效率明显高于对照组细胞。结论联合使用细胞因子bFGF、EGF和PDGF－BB可促进小鼠间BMSCs的体外增殖和干性的维持，并且有利于其向成骨细胞的分化。
Objective To investigate the effects of cytokine factors basic fibroblast growth factor （bFGF）, epidermal growth factor （EGF） and platelet derived growth factor-BB （PDGF-BB） on prolification and osteogenesis of murine mesenchymal stem cells obtained from bone marrow （BMSCs）. Methods Cell adhesion was used to purify the BMSCs in the presense or absence of bFGF, EGF and PDGF-BB （ 10μg,/L） in culture medium composed of DMEM/F12, 10% FBS, and penicillin/streptomycin （100 U/ml and 100 mg/L, respectively）. Cells at the third passages were used for subsequent experiments. Flow cytometry was applied to detect the expression of cell-specific surface antigens CIM5 and CD90. Osteogenic differentiation of BMSCs was measured by osteoblast-specific genes expression of collagen I and osteocalcin, alkaline phosphatase assays and deposition of calcium mineralization. Results Cells in the absence of cytokines showed much faster adhesion rate than in the presence of cytokines after initial culture, but slower proliferative ability after passage, and displayed more senescense form. The expression of CD45 and CD90 in cells in the presence or absence of cytokines was [ （ 0.48 ± 0. 12 ） %, （ 82. 78± 0. 25 ） % ] and [ （27.34 ± 0. 69）%, （41.93 ± 0. 34）%], respectively. BMSCs in the presence of cytokines exhibited higher expression of osteoblast-specific genes, and better formation of calcium mineralization than control groups, indicating cells in the presence of cytokines possessed high potency for osteogenic differentiation. Conclusion Combination of three cytokines could improve the proliferation of mesenchymal stem cells in vitro and facilitate osteogenic differentiation.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2013年第1期122-124,共3页
Chinese Journal of Experimental Surgery
基金
浙江省自然科学基金资助项目（Y2091180）
湖州市科技攻关计划资助项目（2009GG10）
关键词
骨髓间充质干细胞
细胞因子
成骨分化
Mesenchymal stem cells
Cytokine
Osteogenic differentiation
