骨形态发生蛋白-2腺病毒表达载体在成骨过程中与血管内皮细胞生长因子关系

Relationship between Ad-BMP-2 and VEGF and their role in the ossification

[目的]探讨骨形态发生蛋白-2腺病毒表达载体(A recombinant adenoviral vector carrying the human BMP-2 gene,Ad-BMP-2)在成骨过程中和血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)相互关系。[方法]应用人骨形态发生蛋白-2腺病毒载体体外感染兔骨髓间质干细胞(bone marrow stem cells,BMSC),感染后观察BMP-2、VEGF和碱性磷酸酶(alkaline phosphatase,ALP)表达及钙结节形成。同时将30μl Ad-BMP-2行裸鼠左后肢肌组织内注射,术后20 d取材,免疫组化和原位杂交法观察BMP-2与VEGF体内表达情况。30μlβ半乳糖苷酶腺病毒载体(Adβgal)右后肢肌内注射作为对照。[结果]BMSC被Ad-BMP-2感染后,向成骨细胞分化,上调VEGF的表达。在体内成骨过程中,BMP-2在软骨细胞、成骨细胞和再生骨组织周围单核细胞表达阳性,VEGF在软骨细胞、成骨细胞和血管内皮细胞均表达阳性。[结论]BMP-2和VEGF两者协调一致,互为促进,完成了骨组织的发生。

[ Objective] To investigate the relationship of the VEGF and BMP-2 in the bone formation induced by the Ad- BMP-2. [Method] Ad-BMP-2 infected rabbit BMSCs were cultured in vitro, then the expression of hBMP-2, VEGF determined by immunohistochemical and in situ hybridization analysis and ALP was determined by enzyme cytochemistry after infection. At 2t days, Alizarin red S staining was used to evaluate calcium-rich deposits by cells transfected in culture. At the same time, 30μl Ad-BMP-2 solution （2×10^10 pfu/ml） was injected into the left hind-limb muscle of the nude rat, and 30μl Ad β gal solution （2 ×10^10 pfu/ml） were injected into the right hind-limb muscle as control. Animals were sacrifaced at 20 days following injection and the expression of BMP-2 and VEGF in the specimens of the injection sites were investigated by immunohistochemistry and situ hybridization method. [ Result] The expression of BMP-2 and ALP in the transduced BMSCs was confirmed after infection. A deeply stained mineralized layer was evident at 21 days in transduced Cells. The expression of VEGF obviously increased in the BMSCs after infection. In vivo experiment, BMP-2 expression was observed in the chondrocytes, osteoblasts and mesenchymal monocytes, correspondingly, VEGF expression was in the chondrocytes, osteoblasts and vascular endothelial cells in the new bone formation. [ Conclusion] Ad-BMP-2 could infect BMSCs with high efficiency and promote the expression Of VEGF. BMP-2 and VEGF work cooperatively in the process of endochondral ossification.