hBMP-2基因转染兔骨髓间充质干细胞的表达及生物学效应的观察

Expression and biological effects of human BMP-2 gene transfectionon rabbit's bone mesenchymal stem cells in vitro

目的:观察人骨形态发生蛋白-2(humanbonemorphogeneticprotein-2,hBMP-2)基因转染兔骨髓间充质干细胞(bonemarrowmesenchymalstemcells,BMSCs)后的表达及表达产物对BMSCs增殖和向成骨细胞分化的影响。方法:利用腺病毒表达载体Adeno-XTM将hBMP-2基因转染兔BMSCs,用免疫组化染色。检测细胞内BMP-2的表达。然后通过MTT法分析其对细胞增殖的影响,并分别通过体外检测Ⅰ型胶原合成和表达情况、碱性磷酸酶染色和钙结节VonKossa染色,观察腺病毒介导hBMP-2基因转染兔BMSCs的成骨分化能力。结果:转基因细胞6周时仍能表达外源性基因。基因表达产物hBMP-2能明显促进BMSCs的增殖以及I型胶原的合成,转染后第14天碱性磷酸酶染色多数细胞为阳性,第21天出现钙结节。结论:hBMP-2基因转染BMSCs后可获得稳定表达,且基因表达产物能促进BMSCs增殖,并诱导其向成骨细胞分化。

Objective To observe the expression of human bone morphogenetic protein-2 （hBMP-2） gene, which is transferred into rabbit＇s bone mesenchymal stem cells （BMSCs） by adenovirus and detect the proliferation and differentiation of rabbit＇s bone mesenchymal stem cells after gene transfection in vitro. Methods BMSCs were transferred with hBMP-2 gene through Adeno-X？ adenoviral expression systems. Then the expression of hBMP-2 gene was detected with immunohistochemical staining. The changes in the cells proliferation were detected with MTT method and the osteogenic potential of cells was analyzed by type Ⅰ collagen assay with immunohistochemistry, alkaline phosphatase（ALP） assay with modified calcium -cobalt staining method, calcium nodes assay with Von Kossa staining and detecting synthesis of osteocalcin （OC）. Results The expression of ectogenesis gene in transferred BMSCs was detectable in 6 weeks. The transgene expression products could stimulate the proliferation of BMSCs cultured in normal conditions and the biosynthesis of type Ⅰ collagen of BMSCs. Most BMSCs were stained positively for 14 days after transfection and the calcified nodes formed 21 days after transfection. Conclusion BMSCs have the potential for stable gene expression and the transgene expression products can induce BMSCs to differentiate into osteoblast and enhance its proliferation.