重组逆转录病毒hVEGF165基因转染对人骨髓基质细胞成骨影响
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Recombinant retrovirus pLXSN/VEGF165 transfection promotes human marrow stroma cells osteogenesis
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    摘要:

    目的:观察重组逆转录病毒人血管内皮生长因子165(retrovirus pLXSN/hVEGF165)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hMSCs,体外扩增纯化后随机分为4组:①retrovirus pLXSN/hVEGF165组:培养液中加入人血管内皮生长因子基因重组逆转录病毒1×1010OPU/ml,孵育24h后换普通培养液继续培养;②retrovirus pLXSN组:培养液中加入逆转录病毒空载体,其余处理与retrovirus pLXSN/hVEGF165组相同;③阳性对照组:培养液中添加地塞米松-抗坏血酸和β-甘油磷酸钠;④空白对照组:不给予特殊处理-各组细胞处理后2周,免疫组化观察VEGF表达,Von Kossa染色检测人hMSCs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活性-结果:经多次换液传代,人hMSCs呈均一梭形形态-处理后retrovirus pLXSN/hVEGF165组与阳性对照组细胞形态逐渐趋于扁平,突起减少-retrovirus pLXSN组和空白对照组形态无明显变化-免疫组化染色见VEGF主要在胞浆内表达,retrovirus pLXSN/hVEGF165组VEGF表达明显强于其他3组-Von Kossa染色retrovirus pLXSN/hVEGF165组与阳性对照组可见大量红色骨胶原结节形成,显著多于retrovirus pLXSN组和空白对照组-retrovirus pLXSN/hVEGF165组与阳性对照组培养上清液碱性磷酸酶活性显著高于retrovirus pLXSN组和空白对照组-结论:基因重组逆转录病毒人血管内皮生长因子转染对人hMSCs成骨能力具有促进作用-

    Abstract:

    Objective:To investigate the osteogeneous effect of recombinant retrovirus pLXSN/VEGF165 transfection on human marrow stroma cells(hMSCs). Methods:hMSCs isolated from health volunteers,cultured and purified in vitro were randomly divided into 4 groups. In retrovirus pLXSN/hVEGF165 group,recombinant retrovirus pLXSN/hVEGF165 was added into culture medium (1×1010 OPU/ml) and incubated for 24 h. In retrovirus pLXSN group,recombinant retrovirus pLXSN was added to take the place of retrovirus pLXSN/hVEGF165. In positive control group,dexamethasone (1 nmol/L),glycerophosphate (10 mmol/L) and ascorbic (50 mg/L) were added into culture medium for 2 weeks. In blank control group,no special treatment was administrated. Two weeks after treatment,VEGF expression were evaluated by immunohistochemistry,calcified nodes of hMSCs in each group were tested by Von Kossa stain 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 retrovirus pLXSN/hVEGF165 group and positive control group gradually showed flat shape,but did not in retrovirus and blank control group. The level of VEGF expression in retrovirus pLXSN/hVEGF165 group were higher than the others,Von Kossa stain demonstrated that red bone collagen notes of hMSCs in retrovirus pLXSN/hVEGF165 group and positive control group were significantly more than that in retrovirus pLXSN and blank control groups. Similarly,ALP of culture medium in retrovirus pLXSN/hVEGF165 group and positive control group were significantly higher than those in retrovirus pLXSN group and blank control group. Conclusion:Recombinant of retrovirus pLXSN/hVEGF165 gene can transfect hMSCs and promote osteogenesis of hMSCs.

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李〓伟,高鸿雁,王建忠,谢祥仁,肖庆方.重组逆转录病毒hVEGF165基因转染对人骨髓基质细胞成骨影响[J].南京医科大学学报(自然科学版),2010,(3):291-294338

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