BMP-9/EPO双基因共转染ADSCs复合nHAC/PLA支架修复兔下颌骨缺损的实验研究

目的：探讨腺病毒介导BMP-9/EPO双基因共转染脂肪干细胞（ADSCs）复合nHAC/PLA支架材料促进体内成骨的可行性。方法：分离培养兔ADSCs，利用重组腺病毒构建携带BMP-9、EPO、BMP-9/EPO基因的表达载体，并转染至兔ADSCs。14 d后，在荧光显微镜下观察各组细胞荧光表达情况，计算病毒转染效率，采用Western免疫印迹检测各组细胞 BMP-9及EPO蛋白的表达。50只大白兔随机分为5组，A组为空白组，B组为单纯材料组，C组为BMP-9/nHAC/PLA组，D组为EPO/nHAC/PLA组，E组为BMP-9/EPO/nHAC/PLA组。制备兔双侧下颌骨缺损模型，按照实验分组，将转染组细胞分别与nHAC/PLA支架复合培养后植入兔下颌骨缺损处，于4、8、12周在缺损处取材进行影像学、组织学检测及扫描电镜观察，比较各组动物下颌骨缺损修复情况。采用 SPSS17.0软件包对结果进行统计学分析。结果：荧光显微镜下观察，BMP-9、EPO及BMP-9/EPO重组腺病毒均能稳定转染 ADSCs，转染效率为 80%～93%。转染后，BMP-9、EPO及BMP-9/EPO基因和蛋白均能稳定表达。转染后的ADSCs与nHAC/PLA支架复合，其骨缺损区修复情况优于对照组，且BMP-9/EPO/nHAC/PLA组成骨情况优于其他各组。扫描电镜下见转染组骨缺损与材料结合修复情况优于对照组，BMP-9/EPO/nHAC/PLA组修复最明显。结论：BMP-9、EPO均可转染ADSCs并稳定表达。与nHAC/PLA支架材料复合后，可显著促进下颌骨缺损的修复，为骨组织修复工程提供了新的选择。

Repair of critical sized mandibular bone defect with nHAC / PLA scaffold combing with rabbit adipose tissue derived mesenchymal stem cells transfected with BMP-9 / EPO gene

PURPOSE: To investigate the effect of recombinant adenovirus-mediated bone morphogenetic protein-9 (BMP-9) and erythropoietin (EPO) genes co-transfection combined with scaffold materials of nHAC/PLA on osteogenesis in vivo. METHODS: ADSCs were isolated with enzyme digestion and adherence method. The recombinant adenovirus-mediated bone morphogenetic protein-9 (BMP-9), erythropoietin (EPO), and BMP-9/EPO genes co-transfection were constructed respectively, and then ADSCs were transfected with recombinant adenovirus. The expression of cell fluorescence was observed under a fluorescence microscope at 14 days, and the transfection efficiency was calculated. Western blot was used to detect the expressions of BMP-9 and EPO proteins at 14 days. 50 New Zealand rabbits were selected, and bone defect was made in bilateral mandibles respectively, and then randomly divided into 5 groups. ADSCs/nHAC/PLA in each group was transplanted into the bilateral bone defects. Rabbits of each group were killed at 4, 8 and 12 weeks after operation respectively, and the healing of the defects was detected by X-ray, histological and scanning electron microscopy (SEM) examination. Statistical analysis was performed using SPSS 17.0 software package. RESULTS: Fluorescence microscope showed that BMP-9 and EPO, BMP-9/EPO recombinant adenovirus could stably transfected ADSCs, with a transfection efficiency of 80%-93%. Gene and protein of BMP-9, EPO as well as BMP-9/EPO in ADSCs could be stably expressed. Osteogenesis in BMP-9/EPO/nHAC/PLA group was significantly better than other groups. SEM indicated that the interface between materials and bone defect was combined closely in BMP-9/EPO/nHAC/PLA group. CONCLUSIONS: Recombinant adenovirus-mediated BMP-9, EPO and BMP-9/EPO genes can transfect ADSCs, which can stably express in ADSCs. ADSCs transfected with recombinant adenovirus-mediated BMP-9/EPO gene combined with nHAC/PLA could improve bone generation in mandibular defect, and provide a possible option for bone tissue engineering.