携带人骨形态发生蛋白2可诱导慢病毒载体转染人脐带间充质干细胞的成骨

In vitro osteogenetic effects of the Tet-On lentiviral vectors carrying human bone morphologic protein 2 transfected human umbilical cord mesenchymal stem cells

目的:观察携带骨形态发生蛋白2的tet-on慢病毒载体对人脐带间充质干细胞转染表达及稳定转染后成骨的影响。方法:取第3代人脐带间充质干细胞分组培养:实验组稳定转染携带骨形态发生蛋白2的tet-on慢病毒载体,并加入10mg/L强力霉素;未加强力霉素组:同实验组但不加强力霉素;空病毒组转染携带绿色荧光蛋白的慢病毒载体;空白对照组未进行任何处理。结果与结论:①骨形态发生蛋白2表达:实验组、未加强力霉素组均有表达,且实验组表达量高于未加强力霉素组(P<0.05);空病毒组、空白对照组未见表达。②碱性磷酸酶染色:实验组可见细胞胞浆中出现大量红色或红棕色颗粒,未加强力霉素组可见少量红色颗粒,实验组碱性磷酸酶活性高于未加强力霉素组(P<0.05);空病毒组、空白对照组未见明显红色颗粒。③茜素红染色:实验组、未加强力霉素组均可见钙结节形成,实验组较未加强力霉素组矿化结节数量多,面积更大;空病毒组、空白对照组未见明显矿化结节形成。表明携带骨形态发生蛋白2的慢病毒载体可稳定转染人脐带间充质干细胞,显著增强其成骨能力。

BACKGROUND：In addition to the advantages of conventional lentiviral vectors,Tet-On lentiviral vectors can regulate the expression of target gene by doxycycline or similar analogue because of insertion of trans-acting factors.OBJECTIVE：To investigate the effects of Tet-On lentiviral vectors carrying human bone morphologic protein 2 on transfection and osteogenesis of human umbilical cord mesenchymal stem cells（hUMSCs）.METHODS：The cultured passage 3 hUMSCs were divided into four groups.In the hUMSC ＋ doxcycline group,cells were stably transfected with Tet-On lentiviral vectors carrying human bone morphologic protein 2 and 10 mg/L doxcycline was added.In the hUMSC group,cells were treated the same as the hUMSC ＋ doxcycline group,with the exception of addition of doxcycline.In the empty virus group,cells were transfected by green fluorescence protein.In the blank control group,cells were not treated.RESULTS AND CONCLUSION：Human bone morphologic protein 2 expression was significantly higher in the hUMSC ＋ doxcycline group than that in the hUMSC group（P 0.05）.Human bone morphologic protein 2 expression was not detected in the empty virus and blank control groups.Alkaline phosphatase staining results showed that a large number of red or brown granules appeared in the cytoplasm in the hUMSC＋ doxcycline group,and there were a few red or brown granules in the cytoplasm in the hUMSC group.Alkaline phosphatase activity was significantly higher in the hUMSC＋ doxcycline group than in the hUMSC group（P 0.05）.Such red or brown granules were not observed in the empty virus and blank control groups.Tubercles developed in the hUMSC ＋ doxcycline group and hUMSC group.The tubercles were more and larger in the hUMSC ＋ doxcycline group compared with the hUMSC group.Tubercles were not observed in the empty virus and blank control groups.These findings suggest that Tet-On lentiviral vectors carrying human bone morphologic protein 2 can stably transfect human umbilical cord mesenchymal stem cells and significantly increase cellular osteogenic capability.