下颌骨牵引成骨过程中基因干预对牵引区转化生长因子β1表达的影响

Effects of gene therapy on the expression of transforming growth factor beta 1 during mandible distraction osteogenesis

背景:局部基因治疗能促进牵引区新骨的生成,但关于基因治疗后对局部生长因子表达的影响目前尚不清楚。目的:观察电穿孔介导的基因治疗对兔下颌骨牵引成骨过程中转化生长因子β1表达的影响。方法:新西兰大白兔双侧下颌骨截骨后3d开始下颌骨牵引,0.8mm/d,连续牵引7d后,随机分为5组,分别在牵引区注射2μg(0.1g/L)重组质粒pIRES-hVEGF165-hBMP2、pIRES-hBMP2、pIRES-hVEGF165、空质粒pIRES及相同剂量的生理盐水。之后施加电穿孔刺激。结果与结论:免疫组织化学染色发现转化生长因子β1主要在细胞胞浆中表达,给药7d时骨端骨细胞、编织骨痂骨细胞、骨痂表面成骨细胞呈转化生长因子β1染色阳性;14d时新生成的编织骨痂骨细胞、骨痂表面成骨细胞、肉芽组织中的间质细胞、单核巨细胞、多核巨细胞转化生长因子β1染色阳性;28d时转化生长因子β1阳性细胞明显减少。其中注射重组质粒pIRES-hVEGF165-hBMP2、pIRES-hBMP2、pIRES-hVEGF165后转化生长因子β1的表达明显多于注射空质粒pIRES及生理盐水(P<0.05或P<0.01)。说明基因治疗能促进转化生长因子β1的表达,促进牵引区细胞基质的形成和新骨生成。

BACKGROUND： Although recent studies have shown that the local gene therapy can promote the formation of new bone in distraction gap, the effect of gene therapy on the expression of local growth factors is unclear. OBJECTIVE： To investigate the effect of electroporation-mediated gene therapy on the expression of transforming growth factor-β1 （TGF-β1） during mandibular distraction osteogenesis in a rabbit model. METHODS： Mandibular distraction of 0.8 mm/d was performed in New-Zealand rabbits at 3 days after bilateral mandibular osteotomy and lasted for 7 days. After the completion of distraction, the rabbits were randomly divided into five groups. 2 μg （0.1 μg/μL） recombinant plasmid pIRES-hVEGF165-hBMP2, pIRES-hBMP2, pIRES-hVEGF165, pIRES and normal saline was injected into the distraction area respectively. After injection, every group employed electroporation. RESULTS AND CONCLUSION： Immunohistochemical staining showed that TGF-β1 was mainly expressed in the cytoplasm. Seven days after transfection, the staining of TGF-β1 was positive on osteocytes of bone edge, osteocytes of woven bone callus and osteoblasts on the surface of bone callus. At 14 days, the expression of TGF-β1 was found on osteocytes of newborn woven bone callus, osteoblasts on the surface of bone callus, stromal cells in granulation tissue, mononuclear giant cells and multinucleated giant cells. At 28 days, the positive expression of TGF-β1 was decreased obviously. Compared with the injection of pIRES and normal saline, the expression of TGF-β1 was significantly higher after the injection of recombinant plasmid pIRES-hVEGF165-hBMP2, pIRES-hBMP2 and pIRES-hVEGF165 （P 0.05 or P 0.01）. It suggests that gene therapy can promote TGF-β1 expression effectively and promote the formation of new bone and cell matrix in distraction gap.