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

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. Subject headings: Bone Marrow; Mesenchymal Stem Cells; Bone Morphogenetic Proteins; Vascular Endothelial Growth Factors; Lentivirus Infections; Cell Proliferation; Tissue Engineering Funding: the National Natural Science Foundation of China, No. 31160199; the Natural Science Foundation of Guangxi Zhuang Autonomous Region, No. 2014GXNSFAA118263; the Scientific Research Project of Guilin Medical University, No. LX2014254 [ABSTRACT FROM AUTHOR]