hBMP-7基因转染对骨髓间充质干细胞增殖和碱性磷酸酶合成的影响

目的探讨逆转录病毒介导的人骨形态发生蛋白7(humanbonemorphogeneticprotein7,hBMP-7)基因转染对兔骨髓间充质干细胞(bonemarrowmesenchymalstemcell,BMSc)增殖和向成骨细胞分化的影响。方法构建hBMP-7逆转录病毒载体,使用含目的基因的病毒液感染BMSc,免疫组织化学方法检测hBMP-7蛋白的表达,MTT法检测细胞增殖能力,使用流式细胞仪检测细胞周期,NPP法检测碱性磷酸酶合成情况。结果经BMP-7基因转染的兔BMSc有hBMP-7的阳性表达,增殖能力无明显改变(P>0.05),但其合成碱性磷酸酶的能力得到显著提高,与空载体病毒液转染、未经转染的BMSc相比差异有显著性(P<0.01)。结论经BMP-7基因转染的BMSc能够表达外源BMP-7,hBMP-7基因转染能够促进体外培养的BMSc向成骨细胞转化,可用于以BMSc为种子细胞的组织工程化骨组织的构建。

The regulatory effect of human bone morphogenetic protein 7 gene transfer on the proliferation and differentiation of rabbit bone marrow mesenchymal stem cells

To detect the proliferation and differentiation of rabbit bone marrow mesenchymal stem cells (BMSc)transferred by retroviral vector carrying human bone morphogenetic protein7(hBMP -7)gene.Methods hBMP -7-expressing replication-deficient retroviral vector(PT-PLNCX 2 -hBMP 7 )was reconstructed using clone technique and recombinant DNA technique.BMSc were infected with the virus granules.The protein of BMP -7gene in transferred cells were determined by immunohistoch-emistry.The proliferativity of the transferred cell were assayed by methabenzthiazuron(MTT)method and flow cytometer.Alkaline phosphatase(ALP)were also detected using enzyme kinetics.Results Cells transferred by PT-PLNCX 2 -hBMP 7 expressed abundant hBMP 7 protein in the cytoplasm.Positive findings were not found in those cells that were not transferred.After infected with virus there were not significant difference of cell proliferation and cell cycle between the cells transferred by hBMP -7or not(P>0.05).ALP activity in transferred cells were increased significantly(P<0.01).Conclusions hBMP -7can be transferred and stably expressed in the cultured rabbit bone marrow stem cells.Proliferation and cell cycle of the transferred cell were not affected.hBMP 7 gene transfer can be used to induce differentiation of BMSc into osteoblast-like cells.