探究慢病毒介导BMP-2及VEGF-165转染山羊骨髓间充质干细胞向软骨方向分化的影响

Effect of Lentivirus Mediated BMP-2 and VEGF-165 Transfection of Goat Bone Marrow Mesenchymal Stem Cells into Cartilage Differentiation

目的:探究慢病毒介导骨形态发生蛋白2(bone morphogenetic protein-2,BMP-2)及血管内皮生长因子-165(vascular endothelial growth factor-165)转染山羊骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)向软骨方向分化的影响。方法:采用全骨髓贴壁法培养原代山羊的BMSCs,传代后在慢病毒的介导下导入目的基因,研究山羊BMSCs作为基因共转移靶细胞的可行性,以及细胞的体外培养条件、增殖情况,转染后向成软骨方向分化等生物学变化。结果:Lv-BMP2-VEGF165组BMP-2 mRNA的表达与BMP-2组无明显差异(P>0.05),VEGF-165 mRNA的表达与VEGF-165组无明显差异(P>0.05);Lv-BMP2-VEGF165转染组OCN mRNA的表达高于单基因转染组(P<0.05);BMP-2和VEGF-165蛋白只有在Lv-BMP2-VEGF165组中高效率表达;Lv-BMP2-VEGF165组OCN蛋白的表达显著高于其他各组(P<0.01),Lv-BMP2-VEGF165转染组碱性磷酸酶活性明显高于其他组。结论:BMP-2、VEGF-165、BMP-2/VEGF-165可成功转入到山羊BMSCs内,并能长期稳定表达,在新骨形成过程中,二者的协同作用能通过促进碱性磷酸酶的生成、骨钙素的合成并使细胞向成软骨方向发展。

Objective:To investigate the effect of lentivirus-mediated BMP-2 and VEGF-165 transfection of goat bone marrow mesenchymal stem cells into cartilage differentiation.Methods:The primary goat bone marrow mesenchymal stem cells were cultured by whole bone marrow adherent method.The target gene was transfected and mediated by virus to study the feasibility of goat BMSCs as the gene co-transfer target cell and the biological changes of cells in vitro,such as culture conditions,proliferation,and differentiation into cartilage after transfection.Results:The expression of BMP-2 mRNA in transfected group was not significantly different from that in BMP-2 group,and the same as the expression of VEGF-165mRNA in transfected group to that of VEGF-165 group.The expression of OCN mRNA in Lv-BMP2-VEGF165 transfection group was higher than that in single gene transfection group(P<0.05),and BMP2 and VEGF165 proteins were only highly expressed in Lv-BMP2-VEGF165 group.The expression of OCN protein in Lv-BMP2-VEGF165 group was significantly higher than that in other groups(P<0.01),and the activity of ALP in Lv-BMP2-VEGF165 transfection group was significantly higher than those of other groups.Conclusion:BMP2,VEGF165,and BMP2/VEGF-165 can be successfully transfected into goat bone marrow mesenchymal stem cells and can be stably expressed for a long time.The synergistic effect of BMP2/VEGF-165 could promote the production of ALP and the synthesis of osteocalcin,and induce the cells into cartilage.