VEGF基因转染结合脱蛋白骨修复股骨头坏死的实验研究

目的:构建pcDNA3.1/VEGF165质粒及联合脱蛋白骨基因治疗早期股骨头缺血性坏死.方法:将含人VEGF165全长cDNA序列的克隆载体pUC18/VEGF165与pcDNA3.1( )载体构建成重组真核表达质粒pcDNA3.1/VEGF165;69只AVNFH造模成功后的新西兰大白兔随机分成3组.第一组,将脱蛋白骨复合pcDNA3.1/VEGF165质粒植入坏死的股骨头内;第二组植入DPB;第三组仅在股骨头内钻一隧道.股骨头标本术后3天,1,2,4,8和16周获取.RT-PCR检测VEGF165mRNA的表达,Western blot检测VEGF165蛋白的表达,组织形态学分析血管发生和股骨头修复.结果:成功构建pcDNA3.1/VEGF165;第一组VEGF165 mRNA和蛋白表达术后1周达到高峰,时间持续超过3周.血管形成术后2,4周增加,骨形成术后2,4和8周增加,与另两组相比差异呈显著性(P＜0.01).结论:VEGF165基因转染可促进局部血管的早期形成,DPB-VEGF复合物可增加骨形成.脱蛋白骨联合VEGF165基因治疗为骨坏死的修复提供了理论基础.

Experimental study of VEGF gene transfer combined with deproteinized bone to repair avascular necrosis of the femoral head

Objective:To reconstruct pcDNA3.1/VEGF165 and combine it with deproteinized bone(DPB) to repair avascular necrosis of the femoral head (AVNFH).Methods:Clone plasmid pUC18/VEGF165 containing the whole cDNA sequence of VEGF165 was combined with pcDNA3.1(+) to reconstruct pcDNA3.1/VEGF165.Sixty-nine AVNFH model New Zealand adult rabbits with a mean weight of 2.8 kg were randomly divided into three groups.In group 1,DPB combined with the reconstructed plasmid pcDNA3.1/VEGF165 was implanted in the drilled channel of the necrotic femoral head.In group 2,only DPB was implanted.In group 3,only channel was drilled without DPB or plasmid implanted.Femoral head specimens were obtained 3 days,1,2,4,and 8 weeks after operation.The expression of vascular endothelial growth factor (VEGF) was examined by RT-PCR,Western blotting techniques.Angiogenesis and repair of the femoral head were observed by histological and histomorphometric analysis.Results:pcDNA3.1/VEGF165 was reconstructed successfully.In group 1,the expression of VEGF was detected in the femoral head implanted by DPB-VEGF compound.VEGF165 mRNA and protein detected went up to apex 1 week postoperatively and lasted more than 3 weeks.The femoral head implanted by DPB-VEGF compound showed a significant difference in vascularization 2 and 4 weeks after operation and a significant difference in bone formation 2,4 and 8 weeks after operation from those in other groups on histomorphometric analysis (P<0.01).Conclusion:Transfection of VEGF165 gene enhances local angiogenesis in early time and DPB-VEGF compound improves bone formation.Deproteinized bone combined with VEGF gene provides a potential method for treatment of osteonecrosis.