腺病毒介导的骨形成蛋白-2基因修饰促进羊骨髓基质细胞成骨分化的实验研究

目的研究腺病毒介导的骨形成蛋白-2(AdBMP-2)基因转染对羊骨髓基质细胞生长、成骨分化的影响。方法抽取成年健康山羊骨髓,以贴壁培养法培养骨髓基质细胞。以AdBMP-2基因转染体外培养的羊骨髓基质细胞,倒置显微镜观察,计算基因转染效率,绘制细胞生长曲线,并行碱性磷酸酶染色及骨钙素定量分析。采用SAS6.2统计软件包对数据进行SNK法分析。结果细胞转染率达70%±3%,转染目的基因的细胞胞体肥大,呈多边形。生长曲线在AdBMP-2基因转染组、LacZ基因转染组和空白对照组之间无显著差别。目的基因转染后12d,碱性磷酸酶阳性染色面积较对照组增加。转染目的基因后6d,骨钙素含量显著增高,与对照组相比有统计学差异(P<0.01)。结论AdBMP-2基因转染促进了体外培养的骨髓基质细胞向成骨细胞表型转化,可望成为颌骨缺损修复的新型种子细胞。

Osteoblastic differentiation of goat bone marrow stromal cells after AdBMP-2 mediated transduction in vitro

PURPOSE: To investigate the effects of adenuvirus mediated transduction with bone murphogenetic protein-2 on proliferation and osteoblastic differentiation of goat bone marrow stromal cells. METHODS: Adult goat bone marrow stromal cells were separated and cultured in vitro and transduced with either AdBMP-2 or AdLacZ gene. The transfer efficiency was calculated under microscopy. Cell proliferation was described by growth curve. Alkaline phosphatase staining and quantitative osteocalcin expression were detected after gene transduction.The data were analyzed by SAS6.2 software package with SNK test. RESULTS: The transfer efficiency reached 70% +/- 3%. No significant difference was found in cell proliferation among 3 groups. Alkaline phosphatase staining was more pronounced and osteocalcin expression was enhanced in AdBMP-2 transfered group.There was significant difference in osteocalcin expression between AdBMP-2 transferred group and the control group (P < 0.01). CONCLUSION: Adenovirus mediated transduction with bone morphogenetic protein-2 enhanced their osteogenic capacity in vitro which might be an alternative cell source for maxillofacial bone regeneration. Supported by National Natural Science Foundation of China (Grant No.30400502), Science and Technology Commission of Shanghai Municipality (Grant No.04dz05601, 05DJ14006, 055407034, 05QMoX14o6) and Research Grant from Shanghai Education Committee (Grant No. 03BC39, 04YQHB081, Y0203).