hBMP-2基因转染骨髓基质细胞及效果检测

The Expression and Transfection of hBMP-2 Gene in Bone Marrow Stramal Cells

目的 通过观察转染hBMP 2基因在骨髓基质细胞生物学性状变化和检测hBMP 2基因在受体细胞中表达水平 ,探讨骨髓基质细胞作为hBMP 2基因受体细胞的可行性。方法 应用脂质体介导的方法将携带hBMP 2基因的重组载体PcDNA3 hBMP2导入体外培养的骨髓基质细胞中 ,用G4 18筛选获得阳性细胞 ,分别应用PCR技术、原位杂交技术和ELISA检测目的基因DNA、mRNA和蛋白质的存在与表达状况 ,并绘制细胞的生长曲线。结果 PCR方法显示实验组可见约 36 0bp的基因条带 ,原位杂交方法显示实验组约有 15～ 2 0 %的阳性细胞 ,ELISA方法显示培养第 1～ 4天时hBMP 2的含量为 37ng/3× 10 5细胞 ,培养第 11～14天和第 2 5～ 2 8天均约 5 4ng/3× 10 5细胞。转染后阳性细胞的倍增时间和生长时间没有因为目的基因的导入而改变 ,与正常培养的细胞相比差异不显著。结论 骨髓基质细胞可以作为hBMP 2基因的受体细胞 ,基因表达可分泌hBMP 2蛋白质 ,可作为骨组织工程学中的种子细胞。

Objective To explore the feasibility of bone marrow stromal cells (MSC) being used as accepted cells of human bone morphogenetic protein-2 (hBMP-2) gene by studying the instantaneous mRNA and protein expression of hBMP-2 in rabbit's MSC, and to make further study on the effects of transfection on biological behavior of these cells. Methods With the help of lipofectamine, the cultured MSCs were transfected with PcDNA3-hBMP2 and the positive cells were screened out by G418. The evidence of successful transfection in these cells could be obtained by PCR,in situ hybridization and ELISA. Results After transfection with PcDNA3-hBMP2, hBMP-2 gene expressed as a 360 bp in MSCs, and its mRNA was found in 15～20% positive cells of MSCs, and the hBMP-2 proteins secreted by the cultured cells were 37mg/3×10 5 cells 1～4 days after transfection, 54ng/3×10 5 cells 11～14 days and 25～28 days after transfection respectively.Conclusion MSC could be used as the receptive cells of hBMP-2 gene and served as seed cells in bone tissue engineering.