Adenovirus-mediated human bone morphogenetic protein 2 gene transfects bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2013.14.003

Abstract

Abstract:

BACKGROUND: Bone marrow mesenchymal stem cells as the seed cells for repair of bone and cartilage trauma and degeneration have been paid increasing attention.
OBJECTIVE: To investigative the effects of human bone morphogenetic protein 2 gene transfection on Sprague-Dawley rat bone marrow mesenchymal stem cells.
METHODS: Sprague-Dawley rat bone marrow mesenchyal stem cells were in vitro isolated, purified and amplified. Adenovirus-mediated human bone morphogenetic protein 2 was transfected into bone marrow mesenchymal stem cells. CD90 and CD45 expression levels were tested by flow cytometry. The successfully packaged virus was transfected into bone marrow mesenchymal stem cells and expression of human bone morphogenetic protein 2 gene was confirmed by enhanced green fluorescent protein expression under the fluorescence microscope. Enzyme linked immunosorbent assay was performed to monitor the expression levels of human bone morphogenetic protein 2 and alkaline phosphatase in mesenchymal stem cells. The effect of human bone morphogenetic protein 2 on the proliferation of bone marrow mesenchymal stem cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.
RESULTS AND CONCLUSION: Bone marrow mesenchymal stem cells were successfully harvested from bone marrow of sprague-dawley rats and identified by flow cytometry. After primary culture for 7-10 days, cultured cells displayed typical fusiform shape and the growth status was like “cobblestones” or “whirlpool” under light microscope and could be differentiated into osteoblast-, adipocyte- and neuron-like cells in vitro. After transfection by human bone morphogenetic protein 2 gene, bone marrow mesenchymal stem cells expressed human bone morphogenetic protein 2 and alkaline phosphatase. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that after transfection by human bone morphogenetic protein 2 gene, bone marrow mesenchymal stem cells exhibited a stronger proliferation capacity (P < 0.05). These findings suggest that human bone morphogenetic protein 2 gene-transfected bone marrow mesenchymal stem cells can successively express high level of human bone morphogenetic protein 2 and alkaline phosphatase, indicating that human bone morphogenetic protein 2 gene can significantly promote the proliferation of bone marrow mesenchymal stem cells.

Key words: stem cells, bone marrow-derived stem cells, adenovirus vector, human bone morphogenetic protein 2, bone marrow mesenchymal stem cells, gene transfection, rats, alkaline phosphatase, MTT assay, gene engineering, seed cells, bone defects, provincial grants-supported paper, stem cell photographs-containing paper

CLC Number:

R394.2

Yin Cheng-hui, Qiu Jun-qin, Zeng Zhao-xun, Chen Zong-xiong. Adenovirus-mediated human bone morphogenetic protein 2 gene transfects bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(14): 2488-2494.

Figures/Tables 9

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