Construction Of Recombinant Adenvoirus Vectors Encoding Angiopoietin-1 Gene And Transfection On Bone Marrow Mesenchymal Stem Cells

Objectives:(1)To construct and identify the recombinant adenovirus vectors encoding rat angiopoietin-1(ang-1)gene.(2)Evaluate the effects of ang-1 gene transfection on proliferation of bone marrow mesenchymal stem cells(BMSCs) in vitro.Methods:(1)The total RNA was isolated from placenta of rat.The ang-1 gene sequence was amplified by RT-PCR and then cloned into shuttle plasmid pAdTrack-CMV and transformed into BJ5183 carrying backbone plasmid pAdEasy-1 to obtain adenovirus plasmid through homologous recombination.The recombinant vector was selected by kanamycin.Identified by digestion with Pac I and PCR,it was amplified broadly in XL 10-Gold competent bacteria and was packaged and amplified in 293 cell.The infectious titer of the recombinant adenovirus was tested by GFP expression.(2)BMSCs were harvested from bone marrow of SD rat by density centrifugation and the cells were tested CD90 and CD45 by flow cytometry.The successfully packaged virus were transfected to BMSCs and expression of ang-1 gene was confirmed by GFP expression under the fluorescence microscope.Western Blot were carried out to monitor the expression of ANG-1 protein in mesenchymal stem cells and finally evaluated the effect of ANG-1 on the proliferation of BMSCs under hypoxia condition in vitro by MTT assay.Resules:(1)The resulting 1534 bp ang-1 gene fragment was confirmed by sequencing and comparing with the sequence of the ang-1 gene sequences reported in GeneBank.After extracting out recombinant plasmid which is isolated from positive transformed clones the pAdEasy-1-ang-1 was also identified to be correct.The virus titre is 2.44×109U/ml.(2)BMSCs were successfully harvested from bone marrow of SD rat and identified by flow cytometry.After primary culture of 3-4 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 cells,adipocyte and neuron-like cells in vitro.After the transfection of pAdEasy-1-ang-1 the expression of ANG-1 protein in BMSCs was detected by Western Blot.MTT assay suggested that ANG-1 protein expressing in the BMSCs have no significant effect on the proliferation of BMSCs(N+ANG-lvs N:p>0.05;H+ANG-lvs H:p>0.05).Conclusion:The recombinant adenovirus named pAdEasy-1-ang-1 carrying ang-1 gene had been constructed successfully and was transfected into BMSCs stably at high efficiency. Moreover the expression of ANG-1 in BMSCs have no significant effect on the proliferation of BMSCs in vitro.