电穿孔介导的基因治疗对下颌骨牵引新骨生成影响的组织形态计量学研究

Histomorphometry of the effect of electroporation mediated gene therapy on new bone in mandibular distraction area

目的:探索电穿孔介导的基因治疗对下颌骨牵引成骨(distraction osteogenesis,DO)过程中牵引间隙新骨生成的影响。方法:采用新西兰大白兔50只,双侧下颌骨截骨安装牵引器后3天开始牵引,每天0.8mm,连续牵引7天后,将实验动物分为5组,每组10只:A组:在牵引区注射2μg(0.1μg/μl)pIRES-hVEGF165-hBMP2;B组:在牵引区注射2μg(0.1μg/μl)pIRES-hBMP2。C组:在牵引区注射2μg(0.1μg/μl)pIRES-hVEGF165;D组:在牵引区注射2μg(0.1μg/μl)空质粒pIRES;E组:在牵引区注射相同剂量的生理盐水。5组实验动物均施加电穿孔刺激。各组分别于固定期第2、4、8周取材行组织学检查和形态计量学分析,测定牵引区新生骨量和新生骨小梁宽度。结果:A、B、C组新生骨量及新生骨小梁宽度明显高于D、E组(P<0.01)。且A组明显高于B、C组,但B、C组,D、E组间无显著性差异。结论:电脉冲介导的pIRES-hVEGF165-hBMP2重组质粒体内转染可能实现成骨与血供的联合重建,使单一生长因子的效应放大,促进牵引区新骨的生成。

Objective To explore the effects of electroporation mediated gene therapy on the new bone formation in distraction area.Methods Fifty New-Zealand rabbits were employed,mandibula distractor were placed after osteotomy were performed bilaterally steotomy.Activation of the device was commenced after 3 days of latency period and proceeds at the rate of 0.8mm per day for 7 days.After the completion of activation,the rabbits were randomly divided into 5 groups： group A： recombinant plasmid 2μg（0.1μg/μl）pIRES-hVEGF165-hBMP2,was injected into the distraction area,after the completion of activation;group B： recombinant plasmid pIRES-hBMP2 was injected into the distraction area;group C： recombinant plasmid pIRES-hVEGF165 was injected into the distraction area;group D： pIRES was injected into the distraction area,and group E： normal saline（NS） was injected into the distraction area.After injection every group employed electroporation.The rabbits were sacrificed and harvest the distraction area tissue for histomorphometry for 2 weeks,4 weeks and 8 weeks after gene transfected.Results Mean new bone volume of the distraction area in group A,B and C were remarkable higher than those of group D and E（P0.01）.And that of group A were remarkable higher than those of group B and C,but there were no difference between group B and C,group D and E.Conclusion Electroporation-mediated transfecting recombinant plasmid pIRES-hVEGF165-hBMP2 could obtain combination of VEGF and BMP,which may be make osteogenesis and angiogenesis implementation at the same time,Further more,it may be magnify the effect of single growth factor,and improve the osteogenesis.