外源性人骨形态发生蛋白-2基因在兔骨髓基质细胞中的稳定表达及其诱导成骨作用

Stable expression of exogenous human bone morphorgenetic protein-2 in rabbit bone marrow stromal cells and its osteoinductive effect

目的:通过观察携带外源性人骨形态发生蛋白-2(humanbonemor-phorgeneticprotein-2,hBMP-2)基因的骨髓基质细胞(bonemarrowstromalcells,MSCs)生物学性状变化和检测MSC分泌hBMP-2的含量,探讨hBMP-2基因在MSC中的稳定表达。方法:应用脂质体介导的方法将携带hBMP-2基因的重组载体PcD-NA3-hBMP2导入体外培养的兔MSC中,用G418筛选获得阳性细胞,应用ELISA检测MSC表达并分泌hBMP-2水平,并绘制细胞的生长曲线。结果:从细胞的培养基中可检测到hBMP-2蛋白质。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无显著性意义。结论:hBMP-2基因在MSC中可获得较高水平的稳定表达并能分泌hBMP-2蛋白质,为骨缺损修复中内源性hBMP-2作用的发挥提供可能。

AIM:To explore the stable expression of human bone morphorgenetic protein 2(hBMP 2) gene in bone marrow stromal cells(MSCs) by investigating the changes of biological characters of MSCs with exogenous hBMP 2 gene and measuring the levels of hBMP 2 secreted by MSCs. METHODS:With the help of lipofectamine,the in vitro cultured rabbit MSCs were transfected with recombinant vector PcDNA3 hBMP2 bearing hBMP 2 and,the positive cells were screened out by G418.The expression of MSCs and secretion of hBMP 2 from MSCs were measured by ELISA,and then the growth curve of MSCs was described. RESULTS:hBMP 2 proteins secreted by MSCs could be examined in the culture medium.After transfection with PcDNA3 hBMP2,the doubling time and growing time of the positive cells did not change since the target gene was transfected into the cells,and transfected positive cells were insignificantly different from the cells cultured under normal condition. CONCLUSION:hBMP 2 can be expressed stably in a high level in MSCs and also secret hBMP 2 proteins,which provides the possibility for the performance of endogenous hBMP 2 in repairing bone defects.