Transfection of Mouse Bone Marrow Mesenchymal Stem Cells with Lipofectamine-mediated Cytosine Deaminase Genes
摘要
BACKGROUND: The particular bystander effect of suicide gene can remarkably kill tumor cells. Meanwhile, it can be used together with radiotherapy as well as immune gene therapy, and overcome the defect of low gene transduction efficiency. Cytosine deaminase (CD) can generate a powerful bystander effect. OBJECTIVE: To observe the effect of a eukaryotic expression plasmid plRES2-AcGFP1-CD mediated by liposome transfected into bone marrow mesenchymal stem cells (BMSCs) and its gene expression. DESIGN, TIME AND SETTING: A cytologic experiment at genetic level was performed at Research and Development Center of Stem Cell and Tissue Engineering, Dalian University of Technology from May to December 2007. MATERIALS: A total of 6 C57BL mice of SPF degree and weighing 18-20g were used for separation and culture of BMSCs. Escherichia coli DH5a was provided by Research and Development Center of Stem Cell and Tissue Engineering, Dalian University of Technology. Lipofectamine(superscript TM) 2000 was provided by Invitrogen, China. METHODS: The DNA plasmids were extracted from transformed Escherichia coli DH5a. plRES2-AcGFP1-CD plasmid was identified by BamHI/XhoI double digestion. The BMSCs derived from mouse femur and tibia were cultured and purified by adhesion method in vitro. Signal cell suspension prepared by BMSCs of the third generation was cultured by adding fluorescence-labeled CD44, CD45, CD90 and CD105 antibodies. BMSCs of the third generation were transfected by Lipofectamine(superscript TM) 2000 mediation. MAIN OUTCOME MEASURES: Identification of recombinant plasmids; the expressions of surface markers on BMSCs were detected by flow cytometry; the expressions of cytosine deaminase gene were observed at 36 and 48 hours after transfection under fluorescent inverted microscope. RESULTS: After agarose gel electrophoresis, a band appeared at 1.0-1.5 kb of the digested products of plRES2-AcGFP1-CD plasmids, and the band was accorded with the length of cytosine deamin