电穿孔介导的基因治疗对兔下颌骨牵引区新生骨骨密度与强度的影响

The effect of electroporation mediated gene therapy on bone mineral density and strength of new formed bone in mandibular distraction gap in rabbit

目的 探索电穿孔介导的基因治疗对下颌骨DO过程中牵引间隙新生骨密度与强度的影响,从而为促进下颌骨DO新骨生成,缩短牵引周期,减少并发症提供新思路.方法 以新西兰大白兔为实验动物模型,于术后3 d开始下颌骨牵引,每天0.8 mm,连续牵引7 d后,将实验动物分为5组.A组：在牵引区注射2 μg（O.1μg/μl）重组质粒pIRES-hVEGF165-hBMP2;B组：在牵引区注射2 μg（0.1μg/μl）重组质粒pIRES-hBMP2;C组：在牵引区注射2 μg（0.1μg/μl）重组质粒pIRES-hVEGF165;D组：在牵引区注射2 μg（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1、2、4、8周行X线及QCT检查.选整个牵张间隙新生骨痂部分为兴趣区,测定骨密度.然后处死动物.取材测量牵引区新生骨的三点抗压强度.结果 A、B、C组新生骨痂密度各时相点新生骨痂密度明显高于D、E组（P〈0.01）.固定2周,A组明显高于各组,但B、c组间比较差异无统计学意义.固定4周,A、B组明显高于C、D、E组（P〈0.01）.固定8周A组明显高于B、c、D、E组（P〈0.01）.B、C组间比较差异无统计学意义,但高于D、E组（P〈0.01）.固定4周,A组新生骨的三点抗压强度明显高于B、C、D、E组（P〈0.01）.固定8周,A组仍明显高于各组（P〈0.01）,且B组也明显高于c、D、E组（P〈0.05）.结论 电脉冲介导的pIRES-hVEGF165-hBMP2重组质粒体内转染可使牵引区获得较满意的骨再生和骨化成熟进程,其新骨骨化、改建过程均超过对照组.提示联合应用BMP与VEGF,可能会实现成骨与血供的联合重建,并且使单一生长因子的效应放大,使骨愈合的速度加快.

Objective To explore the effect of electroporation mediated gene therapy on bone mineral density and strength of new-formed bone in mandibular distraction gap, so as to enhance the osteogenesis and shorten the distraction term. Methods New-Zeland rabbits were employed. The distraction began after 3 days of latency period at the rate of 0. 8 mm per day for 7 days. After distraction, the rabbits were randomly divided into 5 groups to receive injection in the distraction gap with recombinant plasmid 2 μg（0. 1μg/μl）pIRES-hVEGF165-hBMP2 in group A, with recombinant plasmid pIRES-hBMP2 in group B, with recombinant plasmid pIRES-hVEGF165 in group C, with pIRES in group D, and with normal saline （NS） in group E. After injection, electroporation was performed in all the groups. After 1 week, 2 weeks, 4 weeks and 8 weeks of consolidation, all the animals underwent X-ray and quantitative computed tomography （QCT）. The new-formed bone in distraction gap was selected as regions of interest （ROI） to measure the bone mineral density（ BMD）. Then the rabbits were sacrificed and the new-formed bone samples were harvested to detect 3-point crushing strength. Results BMD of newly formed bone in group A, B and C was markedly higher than that in group D and E （P 〈 0. 01 ）. After 2 weeks of consolidation, BMD in group A was much higher than that in the other groups, but there was no difference between group B and C. After 4 weeks of consolidation, BMD in group A and B was markedly higher than that in group C, D and E （ P 〈 0. 01 ）. After 8 weeks of consolidation, BMD in group A was markedly higher than that in the other groups. While the BMD was not significantly different between group B and C, but the BMD in group B and C was higher than that in group D and E （ P 〈 0. 01 ）. After 4 weeks of consolidation, the 3-point crushing strength of newly formed bone in group A was markedly higher than that in group B,C, D and E （ P 〈 0. 01 ） , which was still the same after 8 weeks of consolidation. And the crushing strength in group B was higher than that in group C, D and E （ P 〈 0.05 ）. Conclusions Electroporation-mediated transfection of recombinant plasmid pIRES-hVEGF165-hBMP2 could greatly enhance osteogenesis and calcification. A combination of VECF and BMP may promote osteogenesis and angiogenesis simultaneously, so as to magnify the effect of each growth factor, resulting a synergetic effect.