慢病毒介导骨形态发生蛋白2和血管内皮生长因子165双基因转染促进骨髓间充质干细胞向成骨细胞分化  

Double transfection with lentivirus-mediated bone morphogenetic protein 2 and vascular endothelial growth factor 165 promotes osteogenesis of bone marrow mesenchymal stem cells

作　　者：周桢杰 李强[1] 李诗鹏 陶旋[1] 马跃刚 Zhou Zhen-jie;Li Qiang;Li Shi-peng;Tao Xuan;Ma Yue-gang(Affiliated Hospital of Guilin Medical University,Guilin 541001,Guangxi Zhuang Autonomous Region,China)

机构地区：[1]桂林医学院附属医院,广西壮族自治区桂林市541001

出　　处：《中国组织工程研究》2018年第25期3950-3955,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金项目(31160199);广西壮族自治区自然科学基金项目(2014GXNSFAA118263);桂林医学院科研项目(LX2014254)。

摘　　要：背景:骨形态发生蛋白2(bone morphogenetic protein 2,BMP-2)和血管内皮生长因子165(vascular endothelial growth factor 165,VEGF-165)均为骨修复过程中必不可少的细胞因子,利用慢病毒转染干细胞研究双因子作用下细胞的生物学改变具有重要意义。目的:探讨慢病毒介导人BMP-2和人VEGF-165双基因转染(2次转染)骨髓间充质干细胞的可行性以及转染后细胞的诱导成骨性能。方法:将骨髓间充质干细胞分为4组:(1)未转染组;(2)空载组:通过2次相同感染复数的空载慢病毒转染细胞;(3)BMP-2组:转染了单个目的基因的细胞(Lv-BMP-2/GFP);(4)BMP-2/VEGF-165组:通过2次转染后携带双目的基因的细胞(Lv-BMP-2/GFP,Lv-VEGH-165/RFP)。转染后不同时间点Western Blot检测目的蛋白表达,MTT法检测各组细胞增殖活性,转染后14 d检测各组细胞碱性磷酸酶活性。结果与结论:(1)空载组、BMP-2组、BMP-2/VEGF-165组在荧光显微镜下均可见相应的绿色及红色荧光蛋白表达;(2)转染后3 d,BMP-2组、BMP-2/VEGF-165组均有相应目的蛋白高表达;转染后7,14,21 d,目的蛋白检测无明显变化(P>0.05);(3)BMP-2/VEGF-165组增殖稍快于BMP-2组(P<0.05),BMP-2组明显快于未转染组、空载组(P<0.05);(4)BMP-2组、BMP-2/VEGF-165组碱性磷酸酶活力明显高于未转染组、空载组;(5)结果表明,慢病毒介导的h BMP-2和h VEGF-165双基因经2次转染成功转入兔骨髓间充质干细胞内,并长期稳定表达,该双因子的表达能通过刺激细胞碱性磷酸酶生成促使细胞更好的向成骨细胞分化。BACKGROUND:Bone morphogenetic protein 2(BMP-2)and vascular endothelial growth factor 165(VEGF-165)are essential cytokines for bone repair.Lentiviral transfection of stem cells is important for studying the biological changes of doubly transfected cells.OBJECTIVE:To study the feasibility of human BMP-2 and human VEGF-165 dual gene transfection of bone marrow mesenchymal stem cells(BMSCs)and to explore the osteogenic capacity of doubly transfected cells.METHODS:Rabbit BMSCs were divided into four groups:untransfected group,blank vector group transfected with control lentivirus,BMP-2 group transfected with Lv-BMP-2/GFP,and BMP-2/VEGF-165 group transfected with Lv-BMP-2/GFP and Lv-VEGH-165/RFP.Expression of targeted proteins was detected using western blot at different time after transfection.MTT assay was used to detect cell proliferation in each group.Alkaline phosphatase activity was measured at 14 days after transfection.RESULTS AND CONCLUSION:(1)The corresponding green and red fluorescent protein expressions were observed under fluorescence microscope in the blank vector,BMP-2,and BMP-2/VEGF-165 groups.(2)The corresponding target proteins were highly expressed in the BMP-2 and BMP-2/VEGF-165 groups at 3 days after transfection,but no significant changes were detected at 7,14,and 21 days after transfection(P>0.05).(3)Compared with the BMP-2 group,the proliferation of cells was slightly faster in the BMP-2/VEGF-165 group(P<0.05),but significantly lower in the untransfected group and blank vector group(P<0.05).(4)The activity of alkaline phosphatase in the BMP-2,and BMP-2/VEGF-165 groups was significantly higher than that in the untransfected and blank vector groups(P<0.05).These findings reveal that BMSCs were successfully transfected by hBMP-2 and hVEGF-165,and the dual gene transfection could promote the production of alkaline phosphatase,thereby accelerating the osteogenic differentiation of BMSCs.

关 键 词：骨髓间充质干细胞 骨形态发生蛋白2 血管内皮生长因子165 慢病毒 基因转染 细胞增殖 碱性磷酸酶 干细胞 国家自然科学基金 骨髓 间质干细胞 骨形态发生蛋白质类 血管内皮生长因子类 慢病毒感染 细胞增殖 组织工程 

分 类 号：R394.2[医药卫生—医学遗传学]