中国口腔颌面外科杂志

中国口腔颌面外科杂志 ›› 2017, Vol. 15 ›› Issue (3): 202-208.doi: 10.19438/j.cjoms.2017.03.003

• 论著 • 上一篇    下一篇

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

张广德1，靳霞1，李荣亮1，杨世茂2，郭延伟1，房殿吉2   

  1. 1.济宁口腔医院 口腔颌面外科，山东 济宁 272000；
    2.济南市口腔医院 口腔颌面外科，山东 济南 250000；
    3.上海闵行区牙病防治所 口腔外科，上海 201100
  • 收稿日期:2016-09-29 修回日期:2016-12-17 出版日期:2017-05-20 发布日期:2017-06-09
  • 通讯作者: 国家自然科学基金（81170935）
  • 作者简介:张广德（1977-），男，主治医师，E-mail：zhangguangde@163.com
  • 基金资助:
    房殿吉，E-mail：dianjifang@yahoo.com.cn

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

ZHANG Guang-de1, JIN Xia1, LI Rong-liang1, YANG Shi-mao2, GUO Yan-wei1, FANG Dian-ji2.   

  1. 1.Department of Oral and Maxillofacial Surgery, Jining Hospital of Stomatology. Jining 272000, Shandong Provence；
    2.Department of Oral and Maxillofacial Surgery, Jinan Hospital of Stomatology. Jinan 250000, Shandong Provence；
    3.Department of Oral Surgery,Dental Institute of Minhang District. Shanghai 201100, China
  • Received:2016-09-29 Revised:2016-12-17 Online:2017-05-20 Published:2017-06-09

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摘要： 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。

关键词: 脂肪干细胞, 腺病毒, BMP-9, 促红细胞生成素, 成骨分化

Abstract: 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.

Key words: ADSCs, Adenovirus, BMP-9, EPO, Osteogenic differentiation

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