重组人骨形成蛋白2基因转染大鼠自体骨髓基质细胞成骨能力研究

目的　在构建的SD大鼠骨组织工程模型中,观察以含孔磷酸钙陶瓷为支架的基因转染自体骨髓基质细胞异位骨形成能力。方法　成年雄性SD大鼠20只,全麻及无菌条件下取其左侧股骨及胫骨,将骨髓冲入 75 ml培养瓶中并在常规培养条件下进行体外扩增;5 d后将部分骨髓基质细胞消化并通过LipofectAMINETM2000介导进行重组人骨形成蛋白(rhBMP2)基因转染及G-418连续筛选14 d;再将转染和未转染的自体细胞一同接种于经纤连蛋白表面修饰的含孔磷酸钙陶瓷上继续培养10 d,并将细胞陶瓷复合体对应地种植于大鼠背部皮下及肌肉内。分别于2~20周处死动物,从形态学及组织学角度观察异位骨形成情况。结果　基因转染细胞陶瓷复合体无论种植于皮下或肌肉内均表现明显的异位骨形成能力;其同期骨形成与早期实验中的经诱导骨髓基质细胞陶瓷复合体异位骨形成相似。结论　应用以含孔磷酸钙陶瓷为支架的rhBMP2基因转染自体骨髓基质细胞移植,可获得与诱导分化的自体骨髓基质细胞相同的成骨能力,说明基因转染自体骨髓基质细胞再次植入体内后,可相当于生物反应器,在增殖的同时促进干细胞的分化和成骨潜能。

Heterotopic Osteogenesis of Autogenous Marrow Stromal Cells with Recombinant Human Bone Morphogenetic Protein 2 Gene Transfection and Porous Calcium Phosphate Ceramic as a Scaffold

OBJECTIVE: To observe the heterotopic osteogenesis of autogenous marrow stromal cells loading on porous calcium phosphate ceramic scaffolds with rhBMP2 gene transfection in a Sprague-Dawley rat model. METHODS: Autogenous marrow stromal cells were obtained from left femurs and tibias of 20 male adult SD rats under general anesthesia and sterile condition and cultured in alpha-Minimal Essential Medium supplemented with 15% fetal bovine serum. RhBMP2 gene was transfected into stromal cells by means of LipofectAMINE 2000 reagent five days after primary culture. The stably gene expressive cells were selected with G-418 for 14 days and mixed with stromal cells without transfection. The mixture cells were seeded and subcultured for another 10 days in porous calcium phosphate bioceramic that had been subjected to surface-modification via soaking in human plasma fibronectin. The cell-ceramic compound was implanted subcutaneously and intramuscularly in the corresponding rat. Lab animals were sacrificed at two-week intervals till twenty weeks postoperatively and the involved samples were removed. RESULTS: Morphologic and histological study demonstrated that cell-ceramic compound had an ability of heterotopic osteogenesis, which was similar to that of autogenous osteoblasts in previous study. CONCLUSION: It seems that autogenous stromal cells with rhBMP2 transfection acts as a bioreactor promoting proliferation and differentiation of stem cells when they are replanted into the corresponding animals.