血管内皮生长因子复合骨形态发生蛋白-2转染骨髓间充质干细胞修复兔股骨头坏死模型

Experimental study of the complex VEGF-BMP-2 transfected BMSCs in repair of osteonecrosis of femoral head of rabbit

目的：探讨血管内皮生长因子（VEGF）联合骨形态发生蛋白（BMP-2）基因转染骨髓间充质干细胞（BMSCs）在治疗兔非创伤性股骨头缺血性坏死（ANFH）中的作用.方法：分离、培养兔BMSCs,采用电转法分别转染重组表达载体pcDNA3.1-BMP-2质粒和重组表达载体pcDNA3.1-VEGF质粒,分为4组,空白组、VEGF组、BMP-2组、混合培养组.免疫蛋白印迹检测各组BMSCs的BMP-2和VEGF蛋白的表达.检测各组碱性磷酸酶（ALP）活性和钙的含量.将64只新西兰大白兔制作ANFH模型,然后随机分为4组,分别转染上述4组质粒,减压后植入ANFH模型,收集股骨头标本分别做X线检查、股骨头组织H-E染色检查.结果：体外实验显示,ALP活性和钙含量,空白组＜VEGF转染组＜BMP-2转染组＜混合转染组.体内实验结果显示第4、8周X线和组织学观察,VEGF和BMP-2真核表达载体联合转染BMSCs组股骨头修复效果优于其他3组.结论：VEGF和BMP-2均能提高BMSCs体外培养中ALP的含量和促进体外培养时BMSCs向骨细胞转化.VEGF复合BMP-2转染BMSCs可增强骨坏死区骨修复和血运重建能力.

Objective： To study the effect of vascular endothelial growth factor （VEGF） combined with bone morphogenetic protein-2 （BMP-2） gene transfection of bone marrow mesenchymal stem cells （BMSCs） on the treatment of early avascular necrosis of femoral head （ANFH） of rabbits. Methods： BMSCs were isolated and cultured according to transfection recombinant expression vector pcDNA3.1-BMP-2 plasmids and recombinant expression vector pcDNA3.1-VEGF plasmid. Models were divided into 4 groups： control group, VEGF eukaryotic expression vector BMSCs transfection, BMP-2 eukaryotic expression vector of BMSCs, mixed VEGF and BMP-2 eukaryotic expression vectors of BMSCs transfection. BMSCs BMP-2 and VEGF protein expression in each group were dectected. Alkaline phosphatase （ALP） activity kits, calcium test kits were used to detect ALP activity and the content of calcium. New Zealand white rabbits were randomly divided into 4 groups. The above four groups of plasmid respectively were transfected into the groups of BMSCs after decompression to repair the defect of ANFH. In each group, femoral head specimens were collected for X-ray, and tissue section and H-E staining to evaluate ANFH repair reconstruction. Results： At 4 and 8 weeks, X-ray and histological observation of VEGF eukaryotic expression vector of transfected BMSCs and BMP-2 eukaryotic expression vector was better than the other three groups. ALP activity and calcium content in control group was less than VEGF transfection group, then BMP-2 group and mixed transfection group. Conclusion VEGF and BMP-2 can improve the content of alkaline phosphatase in BMSCs to promote the transformation of BMSCs into bone cells in vitro culture. Compound VEGF and BMP-2 transfection of BMSCs can strengthen osteonecrosis bone repair and revascularization.