转基因骨髓基质细胞修复骨缺损

Transgenic bone marrow stromal cells in repair of bone defects

背景：转基因细胞移植可以提高成骨细胞数量及成骨活性，有可能成为骨缺损的重要修复方法。目的：构建人骨形态发生蛋白2真核表达载体，鉴定并转染兔骨髓基质细胞，应用基因治疗的方法修复兔桡骨缺损，观察其成骨效果。设计、时间及地点：单一样本观察及同体对照动物实验，于2004-06/2006-02在哈尔滨医科大学附属第一医院中心实验室完成。材料：克隆质粒psp-65BMP2为解放军第四军医大学171腔医院惠赠；pcDNA3．1表达载体购自Invitrogen公司；清洁级新西兰白兔32只，雌雄不限，体质量为2～2．5kg。方法：双酶切克隆载体将骨形态发生蛋白2目的基因与真核表达载体pcDNA3．1连接。将自体骨髓基质细胞修复兔桡骨缺损。分别于16只新西兰白兔缺损处植入载有转基因细胞的胶原，对侧植入单纯胶原对照。16只新西兰白兔缺损处植入载有自体细胞的胶原，对侧植入单纯胶原对照。主要观察指标：①免疫组织化学及Western blotting检测转染骨形态发生蛋白2基因骨髓基质细胞的表达效果。②术后12周麻醉后处死动物，行缺损处大体观察、X射线观察及组织学和电镜观察。结果：①成功构建骨形态发生蛋白2真核表达载体，转染骨髓基质细胞后，免疫组织化学染色，可在骨髓基质细胞胞浆显示棕色阳性染色：Western blotting检测有针对骨形态发生蛋白2抗体的蛋白条带，证实骨形态发生蛋白2表达。②组织学和电镜观察实验侧骨缺损处可见大量成骨细胞及新生基质。对照侧仅在断端间见少量骨细胞，断端处为纤维组织充填：转基因骨髓基质细胞修复骨缺损完全愈合：大体及X射线观察转染实验侧有连续骨痂通过骨缺损处，对照侧无连续骨痂通过断端。结论：骨髓基质细胞经基因转染后可以表达骨形态发生蛋白2，并可以修复骨缺损。

BACKGROUND： Transgenic cell transplantation can elevate quantity and activity of osteoblasts, and can be a major method to repair bone defects. OBJECTIVE： To construct the eukaryotic expression vector of human bone morphogenetic protein 2 （BMP-2）, and to identify and transfect into rabbit bone marrow stromal cells （BMSCs）. To repair rabbit radial defects by gene therapy and to observe osteogenic outcomes. DESIGN, TIME AND SETTING： The single sample and homobody control animal experiment was performed at the Central Laboratory of First Affiliated Hospital of Harbin Medical University between June 2004 to February 2006. MATERIALS： Plasmid DNA psp-65BMP2 （presented by the Hospital of Stomatology, Fourth Military Medical University of Chinese PLA）, plasmid pdDNA3.1（Invitrogen, USA）, 32 New Zealand white rabbits, male or female, weighing 2-2.5kg were used in this study. METHODS： Clone plasmid DNA pspBMP-2 was digested by Xba Ⅰ and Sal Ⅰ, recombined with plasmid pcDNA3.1, and the plasmid was transfected into rabbit BMSCs to repair rabbit radial defect. Transgenic and un-transgenic BMSCs mixing with collagen were separately implanted into 16 bone defect regions of New Zealand rabbits, and another lateral were implanted with collagen only as controls. MAIN OUTCOME MEASURES： Immunohistochemistry and Western blotting of BMSC expression transfected with BMP-2; After anesthesia, animals were sacrificed 12 weeks after surgery, received general observation, X-ray examination, histology test and electron microscope inspection. RESULTS： Eukaryotic expression vector of BMP-2 was successfully constructed. After immunohistochemical staining, the cytoplasm of transfected BMSCs was colored brown. We got white ribbon corresponded with BMP-2 in Western blotting, which identified BMP-2 expression. In histological and electron microscope examination, a large number of osteoblasts and fresh matrix could be seen in experimental sides, but in control sides, there were only few osteoblasts and the gap was filled with fibrous tissues. The bone defect in management of transgenic BMSCs healed completely. The general observation and X-ray result showed bridge callus through bone defect site in experimental sides, whereas no bridge callus in control sides. CONCLUSION： Transfected BMSCs can express BMP-2 and cure bone defect.