Experimental study of heterotopic bone formation by in vivo and in vitro gene transfer of bone morphogenetic protein(BMP) and sonic hedgehog(SHH) in the maxillofacial resions.

从人和小鼠Sonic Hedgehog(Shh)全长cDNA中扩增出编码人和小鼠Sonic Hedgehog(Shh)NH2末端结构域的基因。将其插入到pCR3.1(+)表达载体的多克隆位点。将人和小鼠BMP-2的完整编码片段插入到pCR3.1(+)表达载体和BMP-4的多克隆位点。用磷酸钙沉淀法分别将人和小鼠BMP-2、BMP-4和Shh基因表达载体导入口腔组织来源的人和小鼠成纤维细胞。分别用小鼠BMP-2、BMP-4和Shh基因表达载体分别导入小鼠成纤维细胞克隆，筛选出阳性克隆。将筛选出的阳性克隆在大规模培养瓶中融合生长，将细胞包被在明胶胶囊中，移植到裸鼠大腿肌肉内。植入后14天，软X射线片和光镜下观察到细胞种植区有一不透射线的区域，如…。软骨内骨化形成的新骨较多。小鼠颈部、肩部和四肢的带血管肌瓣被提升以检测其长期存活能力，结果表明胸膜外肌瓣、臀浅肌瓣和股二头肌肌瓣适用于基因转移和细胞植入实验。在A9细胞、C3H10T1/2细胞、KMS-6细胞和来自小鼠口腔组织的成纤维细胞中，分别转染小鼠BMP-2、BMP-4和Shh c DNA载体，在C3H10T1/2细胞和来自小鼠口腔组织的成纤维细胞中检测到BMP和Shh阳性细胞。将转BMP-2、BMP-4和Shh cdna载体的强阳性细胞分别包裹在明胶胶囊中，植入胸外肌皮瓣、臀浅肌瓣和股二头肌皮瓣内，在软片上仅观察到细胞种植区不透射线的区域，光镜检查发现有新的骨形成。将含有BMP-2、BMP-4和Shh cDNA载体的优化液(200μL)分别注射到胸大肌皮瓣内，臀浅肌瓣和股二头肌肌瓣超声2min(2.5W/cm2)。注射后14d，软X线片上仅在溶液注射区观察到一小块不透射线的区域，光镜下可见新生骨形成。较少

cDNA encoding the NH2-terminal domain of human and mouse sonic hedgehog(Shh) was amplified by PCR from the full length human and mouse Shh cDNA. It was inserted into the multiple cloning site of the pCR3.1(+) expression vector. The complete coding resion of human and mouse BMP-2 was inserted into the multiple cloning site of the pCR3.1(+) expression vector as well as BMP-4. Human and mouse fibroblasts derived from oral tissue were transfected with human and mouse BMP-2, BMP-4, and Shh cDNA plasmid vector each by calcium phosphate precipitation. Selected storongly positive clones were mouse fibroblast-clones by transfected with mouse BMP-2, BMP-4, and Shh cDNA plasmid vector each. Selected positive clone was grown to confluence in large scale flasks and cells were enclosed in gelatin capsules and implanted into the thigh muscles of nude mice. 14days after implantation, a radiopaque area was observed in cells-implanted area on the soft X-ray films and light microscopic examination disclo … More sed new bone formation by endochondral ossification. with toluidine bulue staining.Vascularized muscle flaps in mice cervical, shoulder and limbs were elevated to examine longterm-viability and the following results were obtained that ectopectoralis flap, superficial gluteal muscle flap and biceps femoris muscle flap were useful for gene-transfer and cells-implant experiments.In the A9 cells, C3H10T1/2 cells, KMS-6 cells and fibroblasts derived from mouse oral tissues transfected with mouse BMP-2, BMP-4 and Shh cDNA plasmid vector each, BMP and Shh-positive cells were detected in C3H10T1/2 cells and fibroblasts derived from mouse oral tissues. Though BMP and Shh protein secretion was stable by 12 passages, these protein secretions and gene expressions were not detected soon and therefore, these gene expressions were transient as we, had expected.Storongly positive cells transfected with mouse BMP-2, BMP4, and Shh cDNA plasmid vector each were enclosed in gelatin capsules and implanted into ectopectoralis flap, superficial gluteal muscle flap and biceps femoris muscle flap and a radiopaque area was observed in only cells-implanted area on the soft X-ray films and light microscopic examination disclosed new bone formation.Optison solution(200μl) containing mouse BMP-2, BMP-4 and Shh cDNA plasmid vector each were injected into ectopectoralis flap, superficial gluteal muscles flap and biceps femoris muscle flap with ultrasound for 2 minutes(2.5 W/cm2). 14days after injection, a small radiopaque area was observed in only solution-injected area on the soft X-ray films and light microscopic examination disclosed new bone formation. Less