BMP9和VEGF联合诱导小鼠间充质干细胞成骨分化的体外研究

Combination of BMP9 and VEGF induces osteogenic differentiation of C3H10T1/2 mouse mesenchymal stem cells in vitro

目的:探讨重组腺病毒介导的骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)和血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)共感染对骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成骨分化的影响。方法:将重组腺病毒介导的BMP9和VEGF分5组感染小鼠间充质干细胞C3H10T1/2,Ad-BMP9组、Ad-VEGF组、AdVEGF+Ad-BMP9组、空载体腺病毒组和空白组,感染7 d后,real-time PCR检测BMP9和VEGF的m RNA水平,real-time PCR检测成骨指标骨钙蛋白(osteocalcin,OC)、骨保护素(osteoprotegerin,OPG)、骨桥蛋白(osteopontin,OPN)的m RNA水平,Western blot检测OPN蛋白的表达,各组细胞行碱性磷酸酶(alkaline phosphatase,ALP)活性的定量检测、ALP染色和钙盐沉积的检测。结果:BMP9和VEGF在Ad-VEGF+Ad-BMP9组高效共表达,OC、OPG、OPN m RNA水平和OPN蛋白水平在Ad-BMP9组和Ad-VEGF+Ad-BMP9组的表达明显高于其他各组(P﹤0.05),且在Ad-VEGF+Ad-BMP9组表达最高(P﹤0.05)。Ad-VEGF+AdBMP9组的ALP活性、ALP染色和钙盐沉积明显高于其他各组(P﹤0.05)。结论:联合VEGF可明显增强BMP9诱导C3H10T1/2细胞成骨分化能力。

Objective：To investigate the effects of recombinant adenovirus-mediated co-infection of bone morphogenetic protein 9（BMP9）and vascular endothelial growth factor（VEGF）gene on osteogenic differentiation of bone marrow mesenchymal stem cells（BMSCs）. Methods：C3H10T1/2 cells were infected by recombinant adenovirus-mediated of BMP9 and VEGF as follow five groups：Ad-BMP9 group,Ad-VEGF group,Ad-VEGF＋Ad-BMP9 group,Ad-null group and control group. After infecting for 7 days,RT-PCR was performed to detect the m RNA expression level of BMP9 and VEGF,and real-time PCR was carried out to detect the m RNA expression level of osteogenesis index osteocalcin（OC）,osteoprotegerin（OPG）,osteopontin（OPN）. Western blot was used to detect the protein expressions level of OPN. Alkaline phosphatase（ALP）activity was measured by quantitative and staining assay,and calcium deposition was determined by Alizarin Red S staining in each group. Results：BMP9 and VEGF co-expressed effectively in Ad-VEGF＋Ad-BMP9 group. The m RNA level of OC,OPG,OPN and the protein level of OPN in Ad-BMP9 group and Ad-VEGF＋Ad-BMP9 group were statistically higher than those of other groups（P0.05）,and were the highest in Ad-VEGF＋Ad-BMP9 group（P0.05）.The activity and staining assay of ALP,and the deposition of calcium in Ad-VEGF ＋Ad-BMP9 group were statistically higher than those in the other groups（P0.05）. Conclusion：Combination of BMP9 and VEGF may promote osteogenic differentiation of C3H10T1/2cells in vitro.