利用包载骨形态发生蛋白-4腺病毒异位成骨移植修补骨缺损的研究  

In vivo study of repairing bone defects with heterotopic bone grafting containing bone morphogenetic protein-4 adenovirus

作　　者：田科[1] 宋瑞鹏[1] 李鹏举 毕方刚[1] 范道洋 王卫东[1] Tian Ke;Song Ruipeng;Li Pengju;Bi Fanggang;Fan Daoyang;Wang Weidong(Department of Orthopedics,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)

机构地区：[1]郑州大学第一附属医院骨科,450052

出　　处：《中华实验外科杂志》2020年第12期2270-2273,共4页Chinese Journal of Experimental Surgery

基　　金：河南省教育厅2019年重点科研项目(32211051);河南省科技厅2020年科技攻关项目(22170139)。

摘　　要：目的构建包载骨形态发生蛋白(BMP)-4的腺病毒并利用其骨骼肌内异位成骨特性,探索移植异位形成的自体骨进行骨缺损修补的体内效果。方法2018年6月至2020年3月,将BMP-4基因构建入AAV载体内,纯化出AAV-BMP-4。将AAV-BMP-4吸附于明胶海绵(Gelfoam)上并植入郑州大学实验动物中心SPF饲养室饲养的SCID小鼠的股肌袋内,4周后进行AAV-BMP-4的活性测定。在小鼠背部皮下应用AAV-BMP-4,观察骨骼肌肉诱导异位成骨,待骨组织形成稳定后,取出这些骨组织移植入事先制备好的小鼠的颅骨缺损模型中,观察其骨缺损修复效果。结果成功构建出纯化后浓度达5×1012 vp/ml的AAV-BMP-4病毒颗粒。病毒颗粒植入小鼠的股肌袋内可成功诱导异位骨组织的形成,小鼠背部皮下植入AAV-BMP-4+Gelfoam复合体后4周出现了少量的新生骨组织,骨量达1 cm×2 cm×2 cm。X线和MicroCT的检查均证实了小鼠的背部皮下骨组织形成。经修剪移植入小鼠颅骨缺损模型处的骨组织在植入缺损处后,没有出现异常增生的现象,而是不断地随着时间的推移进行着外形的轻微改建,颅骨缺损得到了良好的修复。结论体外构建的AAV-BMP-4具有良好的生物学活性,AAV-BMP-4+Gelfoam在骨骼肌肉中可有效诱导成骨,利用新生骨组织可以成功地修复颅骨缺损的动物模型。移植的骨组织植入颅骨缺损后未出现过度生长的现象,且与宿主骨结合良好。Objective To construct an adenovirus containing bone morphogenetic protein(BMP)-4 and use its characteristic of ectopic bone formation in skeletal muscle to explore the in vivo effect of transplanting heterotopic autologous bone for bone defect repair.Methods The BMP4 gene was constructed into AAV vector,and AAV-BMP4 was purified.AAV-BMP4 was adsorbed in Gelfoam and implanted into the femoral muscle pocket of SCID mice raised in the SPF breeding room of the Experimental Animal Center of Zhengzhou University.The activity of AAV-BMP4 was determined 4 weeks post operation.Apply AAV-BMP4 subcutaneously on the back of the mouse to observe the skeletal muscle induced ectopic bone formation.While the bone tissue is stable,the bone tissue is taken out and transplanted into the pre-prepared mouse skull defect model to observe the bone defect repair effect.Results The purified AAV-BMP-4 virus particles with a concentration of 5×10^12 vp/ml were successfully constructed.The purified AAV-BMP-4 virus particles implanted into the femoral muscle pocket of mice can successfully induce the formation of ectopic bone tissue.A small amount of new bone tissue appeared 4 weeks after the AAV-BMP-4+Gelfoam complex was implanted subcutaneously on the back of the mouse with an amount of 1 cm×2 cm×2 cm.Both X-ray and MicroCT examinations confirmed the formation of subcutaneous bone tissue on the back of the mouse.The bone tissue that was trimmed and transplanted into the mouse skull defect model did not show abnormal proliferation after being implanted in the defect,but continued to undergo slight changes in appearance over time,and the skull defect was well repaired.Conclusion The AAV-BMP-4 constructed in vitro has good biological activity.AAV-BMP-4+Gelfoam can effectively induce bone formation in skeletal muscle.The new bone tissue can successfully repair the animal model of skull defect.After the transplanted bone tissue was implanted into the skull defect,there was no overgrowth,and it was well integrated with the host bone.

关 键 词：腺病毒 AAV-骨形态发生蛋白-4 诱导分化 骨缺损 成骨 

分 类 号：R687.3[医药卫生—骨科学]