Effects Of Cxcr4 Gene Transfection On The Capacity Of Bone Marrow Mesenchymal Stem Cells Migration To SDF-1

Objectives:(1)To construct an adenovirus vector encoding mouse cxcr4 gene;(2) To study effects of cxcr4 gene modification on the CXCR4 expression mouse BMSCs;(3) To investigate their capacity of migration to SDF-1 in vitro.Methods:(1)BMSCs were harvested from bone marrow of C57BL/6 mice by density centrifugation and the cells were tested CD44 and CD34 by flow cytometry.(2)The cxcr4 gene sequence was amplified by RT-PCR and then cloned into shuttle plasmid pAdTrack-CMV and transformed into Escherichia coli BJ5183 carrying backbone plasmid pAdeasy-1 to obtain adenovirus plasmid through homologous recombination.The adenovirus plasmid was transfected into HEK293 cells to form adenovirus particle.The successfully packaged virus was upscaled through 3 to 5 circles and then its titer was determined.(3)Transfect BMSCs and cxcr4 expression was confirmed by GFP expression and western blotting.(4)Chemotaxis assays were performed to evaluate the migratory capacity of BMSCs to SDF-1 in vitro.For the blocking assay,CXCR4 blocking antibody was added into cell culture.Results:(1) BMSCs can be isolated,cultured and identificated.(2)After extracting out recombinant plasmid which is isolated from positive transformed clones,the sequence of cloned mouse cxcr4 gene was proved to be correct.The virus titre is 5.3×10¹⁰U/ml.(3)The expression of CXCR4 in normal BMSCs is low but were significantly increased after transfection of cxcr4.(4) In the Transwell cell migration test,the directional migration of transfected BMSCs to SDF-1 was improved significantly than untransfected BMSCs(P＜0.05).Conelusion:Adenovirus vector encoding mouse cxcr4 gene transfer into mouse BMSCs can enhanced expression of CXCR4 in mouse BMSCs and the migratory capacity of BMSCs to SDF-1 was enhanced as well.