腺相关病毒介导bFGF基因转染促进人骨髓基质细胞成骨实验研究

THE STUDY OF ADENO-ASSOCIATED VIRUS MEDIATED BASIC FIBROBLAST GROWTH FACTOR TRANSFECTION PROMOTES HUMAN MARROW STROMA CELLS OSTEOGENESIS

目的:观察腺相关病毒介导人碱性成纤维细胞因子(bFGF)基因转染对人骨髓基质细胞(hMSCs)成骨能力的影响。方法:从健康志愿者全骨髓中分离培养人骨髓基质细胞,体外扩增纯化后随机分为4组。①腺相关病毒介导人碱性成纤维细胞因子(AAV-bFGF)组:培养液中加入人碱性成纤维细胞因子基因重组腺相关病毒1×1010OPU/ml,孵育24h后换普通培养液继续培养;②β半乳糖酐酶基因重组腺相关病毒(AAV-LacZ)组:用半乳糖酐酶基因重组腺相关病毒替换bFGF,其余处理与AAV-bFGF组相同;③阳性对照组:培养液中添加地塞米松(1nmol/L)、抗坏血酸(10mmol/L)和β-甘油磷酸钠(50mg/L);④空白对照组:不给予特殊处理。各组细胞处理后2wk,免疫组化观察bFGF表达,Von Kossa染色检测人骨髓基质细胞中骨胶原结节形成,全自动生化分析仪检测培养上清碱性磷酸酶(ALP)活性。结果:经多次换液传代,人骨髓基质细胞呈均一梭形形态。处理后AAV-bFGF组与阳性对照组细胞形态逐渐趋于扁平,突起减少。AAV-LacZ组和空白对照组形态无明显变化。免疫组化染色见bFGF主要在胞浆内表达,AAV-bFGF组bFGF表达明显强于其他3组。von Kossa染色AAV-bFGF组与阳性对照组可见大量红色骨胶原结节形成,显著多于AAV-LacZ组和空白对照组。培养上清液ALP活性检测AAV-bFGF组与阳性对照组显著高于AAV-LacZ组和空白对照组。结论:基因重组腺相关病毒人碱性成纤维细胞因子转染对人骨髓基质细胞成骨能力具有促进作用。

objective：To investigate the osteogenesis effect of Adeno-associated virus mediating basic fibroblast growth factor（AAV-bFGF） genetic transfection on human marrow stroma cells（hMSCs）.Methods：hMSCs were isolated from health volunteers,cultured and purified in vitro,and randomly divided into 4 groups.①AAV-bFGF group,bFGF recombinant Adenovirus-associated virus was added into culture medium（1 ×1010OPU/mL） and incubated for 24h.②AAV-LacZ group,AAV-bFGF was replaced by LacZ recombinant Adenovirus-associated virus,others were same as AAV-bFGF group.③positive control group,dexamethasone（1nmol/L）,glycerophosphate（10mmol/L） and ascorbic（50mg/L） were added into culture medium for 2wk.④control group,no special treatment was Administrated.Two weeks after treatment,bFGF expression were evaluated by immunohistochemistry,calcified nodes of hMSCs in each group were tested by Von Kossa staining and alkaline phosphatase（ALP） of culture medium in each group was detected.Results：hMSCs were uniform spindle-shape fibroblast after several passages.After treatment,hMSCs in AAV-bFGF group and in positive control group gradually showed flat shape,but did not In AAV-LacZ and blank control group.In immunohistochemistry,the level of bFGF expression in AAV-bFGF group were higher than the others,Von Kossa staining demonstrated that red bone collagen notes of hMSCs in AAV-bFGF group and positive control group were significantly more than in AAV-LacZ and blank control group.Similarly,ALP of culture medium in AAV-bFGF group and in positive control group were significantly higher than in AAV-LacZ group and blank control group.Conclusions：Adeno-associated virus mediated bFGF gene can transfect hMSCs and promote osteogenesis of hMSCs.