局部基因治疗对兔新生骨痂中Wnt3a和β-连环蛋白表达的影响

Effect of local gene therapy on expression of Wnt3a andβ-catenin in callus of distraction gap during mandibular distraction osteogenesis in rabbits

目的探讨电穿孔介导的局部基因治疗对兔下颌骨牵引区新生骨痂中Wnt3a和β-连环蛋白(catenin)表达的影响。方法2019年9—12月,在南京医科大学友谊整形外科医院实验室进行实验,新西兰大白兔48只分为对照组、基因治疗组、生理盐水组,每组16只。3组动物双侧下颌骨截骨后4 d以0.8 mm/d速度行下颌骨牵引,连续牵引7 d进入固定期。对照组只行牵引;基因治疗组在牵引开始时在牵引区局部注射重组质粒pIRES-hBMP2-hVEGF165,并给予电穿孔刺激;生理盐水组在牵引开始时,在牵引区局部注射与基因治疗组相同剂量的生理盐水后,施加电穿孔刺激。3组分别于牵引结束、固定7、14、28 d取牵引区新生骨骨痂行免疫组织化学染色和RT-PCR检查Wnt3a和β-catenin表达。结果免疫组织化学染色显示,Wnt3a和β-catenin主要定位于骨痂组织的成纤维细胞、软骨细胞、成骨细胞胞质和胞核中。免疫组织化学和RT-PCR检查结果显示,Wnt3a和β-catenin表达在牵引结束时达高峰,随着牵引张力的消失,逐渐降低。经基因治疗干预后,Wnt3a和β-catenin基因及蛋白表达显著升高,与对照组和生理盐水组比较,基因治疗组在各时点Wnt3a、β-catenin表达量最高,差异有统计学意义(F=96.3,P<0.01)。结论基因治疗促进牵引区新生骨痂Wnt3a和β-catenin表达,以此调节骨重建系统的平衡,从而促进牵引区新骨生成。

Objective To investigate the effect of electroporation-mediated local gene therapy on the expression of Wnt3a andβ-catenin in callus of distraction gap during mandibular distraction osteogenesis of rabbits.Methods The experiments were conducted in the laboratory of the Affiliated Friendship Plastic Surgery Hospital of Nanjing Medical University from September 2019 to December 2019.Forty eight New-Zealand rabbits were randomly divided into control group(group A),gene therapy group(group B)and normal saline group(group C),with 16 rabbits in each group.After bilateral mandible osteotomy and distractors were implanted,the distractors were activated at a speed of 0.8 mm/d on 4th day,postoperatively,and lasted for 7 days,followed by consolidation period.Group A distracted only,group B was subject to local injection of recombinant plasmid pIRES-hBMP2-hVEGF165 in the distraction gap and electroporation stimulation at the beginning of activation distractors;and group C local injection of the same dose of normal saline in the distraction gap and electroporation stimulation at the beginning of activation distractors.Four animals in each group were sacrificed on the day at the end of distraction,7th,14th,28th days of consolidation period,respectively.The callus in the distraction gap was taken for immunohistochemical staining and RT-PCR to detect the expression of Wnt3a andβ-catenin,and image analysis was performed.SPSS 22.0 statistical software was used for data analysis.Results Immunohistochemical staining showed that Wnt3a andβ-catenin were mainly located in the cytoplasm and nuclei of fibroblasts,chondrocytes and osteoblasts in callus tissue.Immunohistochemistry and RT-PCR showed that the expression of Wnt3a andβ-catenin reached a peak at the end of distraction.With the disappearance of distraction tension,the expression of Wnt3a andβ-catenin gradually decreased.After gene therapy intervention,the expression of Wnt3a andβ-catenin was significantly increased,and the expression of Wnt3a andβ-catenin in group B was the highest at each time point,with statistically significant difference compared with groups A and C(F=96.3,P<0.01).Conclusions Gene therapy promotes the expression of Wnt3a andβ-catenin in the callus of distraction gap,regulating the balance of the bone reconstruction system and thus promoting the formation of new bone in the distraction gap.