腺病毒介导骨形态发生蛋白-2基因转移促进人间充质干细胞成骨化.docVIP

腺病毒介导骨形态发生蛋白?2基因转移促进人间充质干细胞成骨化 　;作者：孙志波， 杨述华， 禹志宏， 王家宁 邹海兵， 张磊;【摘要】; 目的:观察腺病毒介导的骨形态发生蛋白?2（BMP?2）基因转移对人间充质干细胞（hMSCs）成骨能力的影响。方法:从健康志愿者全骨髓中分离培养hMSCs，体外扩增纯化后随机分为4组。(1)Ad?BMP?2组：培养液中加入BMP?2基因重组腺病毒（Ad?BMP?2，1times;1010OPU/mL）孵育24 h；(2)Ad?LacZ组：培养液中加入半乳糖酐酶基因重组腺病毒（Ad?LacZ）；(3)阳性对照组：培养液中添加地塞米松、抗坏血酸和beta;?甘油磷酸钠。(4)空白对照组。2 w后行Von Kossa染色检测hMSCs中骨胶原结节形成；生化分析仪检测碱性磷酸酶（ALP）活性。结果:经多次换液传代，hMSCs呈均一梭形。Ad?BMP?2组与阳性对照组细胞形态逐渐趋于扁平，突起减少，Von Kossa染可见大量红色骨胶原结节形成，ALP活性也显著增高。结论:Ad?BMP?2基因转染对hMSCs成骨能力具有促进作用。 【关键词】; 间充质干细胞；骨形态发生蛋白；腺病毒；基因转移；骨生成 ; Abstract：Objective To investigate the effect of adenovirus mediated bone morphogenetic protein 2 (Ad?BMP?2) gene transfer on human osteogenesis of mesenchymal stem cells (hMSCs).Methods hMSCs were isolated from healthy volunteers,cultured and purified in vitro,and randomly divided into 4 groups.In Ad?BMP?2 group,BMP?2 recombinant adenovirus (Ad?BMP?2) was added into culture medium(1times;1010OPU/mL) and incubated for 24 h.In Ad?LacZ group,Ad?LacZ was used with the same protocol as Ad?BMP?2 group.In positive control group,dexamethasone (1nmol/L),glycerophosphate (10 mmol/L) and ascorbic acid (50 mg/L) were added into culture medium for 2 weeks.In blank control group,no special treatment was given.Two weeks after treatment,calcified nodes of hMSCs were tested by Von Kossa staining and alkaline phosphatase (ALP) in culture medium was detected.Results hMSCs were uniform spindle?shaped fibroblast after several passages.After treatment,hMSCs in Ad?BMP?2 group and in positive control group gradually showed flat shape.Von Kossa staining demonstrated that red bone collagen nodes of hMSCs in Ad?BMP?2 group and positive control group were significantly more than in Ad?LacZ and blank control group.ALP in culture medium in Ad?BMP?2 group and in positive control group were significantly higher than in Ad?LacZ group and blank control group.Conclusion Adenovirus mediated BMP?2 gene transfer may promote osteogenesis of hMSCs． ; Key words:Mesenchymal stem cells;Bone mo