人骨形成蛋白4基因修饰的兔骨髓基质细胞异位成骨试验

Ectopic Study of hBMP4 Gene Modified Bone Marrow Stromal Cells in Rabbits

目的:通过人骨形成蛋白4(bonemorphogeneticprotein,BMP4)基因修饰的兔骨髓基质细胞(bonemarrowStromalcells,bMSCs)与天然型无机骨(naturalnon鄄organicbone,NNB)支架复合,进行自体皮下异位成骨试验,探索BMP4基因治疗与组织工程技术相结合的可行性。方法:贴壁法培养兔bMSCs,体外应用脂质体介导转染pEGFP鄄hBMP4及pEGFP真核表达质粒。将转染基因的细胞和未转染基因的对照组细胞,分别以5×107/ml的浓度,与NNB支架复合,构建组织工程化骨,植入6只新西兰兔自体皮下,每组6例,4周后取材,组织学观察,并进行新骨成骨面积分析。结果:空白对照NNB孔隙内无新骨形成,转染pEGFP基因组及对照细胞组,支架网孔内均有新生骨及软骨样组织形成,而pEGFP鄄hBMP4实验组形成的新生骨及软骨样组织面积更大(P<0.05)。结论:应用hBMP4基因修饰的bMSCs作为骨组织工程的种子细胞,可望取得更强的成骨能力。

Objective: hBMP-4 gene therapy combined with tissue-engineering techniques was explored to improve osteogenesis in an ectopic bone formation model in rabbits. Methods: Autologous bone marrow stromal cells (bMSCs) obtained from rabbits were cultured and transfected with either pEGFP-hBMP-4,pEGFP by lipofectamine or left uninfected in vitro. The cells of the 3 groups were combined with Natural Non-organic Bone (NNB) at a concentration of 5×107 cells/ml to construct tissue-engineered bones respectively. They were further implanted into 6 rabbits subcutaneously using NNB as blank control (6 implants per group). Four weeks after surgery, the implants were evaluated with histological staining and computerized analysis of new bone formation. Results: In the ectopic bone formation model,NNB alone could not induce new bone formation. The new bone area formed in the untransfected bMSCs group was 17.2±7.1%, and pEGFP group was 14.7±6.1%, while pEGFP-BMP4 group was 29.5±8.2%,which was the highest among the groups (P<0.05). Conclusion: hBMP4 gene modified bMSCs increased ectopic new bone formation in rabbits. These results indicated that the transfection of bMSCs with hBMP-4 expression plasmid might be suitable for bone tissue engineering applications.