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

doi:10.3969/j.issn.2095-4344.0954

Abstract

Abstract:

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.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Bone Marrow, Mesenchymal Stem Cells, Bone Morphogenetic Proteins, Vascular Endothelial Growth Factors, Lentivirus Infections, Cell Proliferation, Tissue Engineering

CLC Number:

R394.2

Zhou Zhen-jie, Li Qiang, Li Shi-peng, Tao Xuan, Ma Yue-gang. Double transfection with lentivirus-mediated bone morphogenetic protein 2 and vascular endothelial growth factor 165 promotes osteogenesis of bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(25): 3950-3955.

Figures/Tables 2

2.1 原代细胞的分离纯化与鉴定结果原代细胞分离后于培养箱内静置48 h，再给予首次换液，镜下可见散在簇状分布的BMSCs，呈短梭形、三角形。随培养时间延长及数次换液后，杂质细胞可被洗去。原代BMSCs培养7 d左右，细胞可达80%-90%融合，为防止细胞老化给予传代培养。传代后细胞分布均匀，排列呈漩涡状、鱼群状，细胞呈短梭形、三角形，椭圆形胞核明显，有的可见双核(图1)。流式细胞仪检测结果显示，培养的第3代BMSCs表面标志物CD44、CD29阳性表达，其阳性率达90%以上，CD45阴性表达，结果符合BMSCs特征。 2.2 原代细胞的双基因转染用携带hBMP-2基因的慢病毒载体以MOI=50转染第3代BMSCs时，转染后未见细胞死亡，转染后第3天将携带hVEGF-165基因的慢病毒载体以MOI=50再次转染，依然未见细胞死亡。首次转染第7天，即第2次转染后第4天，BMP-2/VEGF-165组和空载组细胞在荧光显微镜下均见到绿色和红色荧光表达，荧光下细胞形态未见明显改变，肉眼计数绿色和红色荧光表达量均可达85%，综合转染率可达95%以上(图2)，说明病毒携带的BMP-2和VEGF-165基因成功转入细胞中。 2.3 转染前后细胞双基因目的蛋白表达 BMP-2/VEGF- 165组及BMP-2组检测到Mr 30 000左右大小的BMP-2阳性条带，而空载组和未转染组在该区域上仅见较弱条带；BMP-2/VEGF-165组在Mr 17 000左右大小区域检测到VEGF-165阳性条带，其余3组在该区域均未见阳性条带，见图3。各时间点灰度值分析见表1。在第7，14，21天时，BMP-2组及BMP-2/VEGF-165组的BMP-2表达差异无显著性意义(P > 0.05)，但均高于第3天，差异有显著性意义(P < 0.05)；在第7，14，21天时，BMP-2/VEGF-165组的VEGF165表达差异无显著性意义(P > 0.05)，但均高于第3天，差异有显著性意义(P < 0.05)。上述结果说明慢病毒介导的BMP-2和VEGF-165基因成功转入细胞内并长期稳定表达目的蛋白，目的蛋白未随细胞增殖传代而表达降低。 2.4 转染前后细胞形态变化转染成功后第7天，可见BMP-2组和BMP-2/VEGF-165组细胞质内有少量颗粒，细胞形态均未见明显改变，呈梭状，胞核明显。转染成功后第14天，BMP-2组和BMP-2/VEGF-165组细胞质内颗粒明显增多，并且后者较前者稍多，两组细胞变宽、变大，伸出伪足，有极性。未转染组和空载组可见极少数细胞胞质内出现质密颗粒，细胞形态保持梭状。 2.5 转染前后细胞增殖能力变化 BMP-2/VEGF-165组BMSCs增殖稍快于BMP-2组(P < 0.05)，后者又明显快于空载组和未转染组(P < 0.05)。未转染组和空载组细胞增殖曲线的走行方向基本一致，差异无显著性意义(P > 0.05)。该结果再次提示该双基因转染方法未对细胞造成损伤，可以排除慢病毒转染带来的干扰，见图4。 2.6 转染前后细胞碱性磷酸酶定性及定量检测结果转染后第14天，BMP-2/VEGF-165组碱性磷酸酶染色可见大部分细胞胞浆被密集的细沙样黑色颗粒所深染，细胞核周围染色最深，显现出明显的未被染色细胞核部分，细胞整体深染率可达90%，这与细胞形态学观察时看到的胞核周围质密颗粒相符，表明这些质密颗粒含大量碱性磷酸酶。BMP-2组细胞染色形态与双基因转染组相似，但整体深染率为80%。未转染组和空载组仅约5%细胞被深染，见图5。转染后第14天各组碱性磷酸酶活力，见图6，4组间两两比较差异均有显著性意义(P < 0.05)，BMP-2/VEGF-165组碱性磷酸酶活性较BMP-2组升高约1/5，但未转染组和空载组比较差异无显著性意义(P > 0.05)。"

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