Culture of endothelial progenitor cells from rabbit peripheral blood and HTK gene modification / 基础医学与临床

ABSTRACT

Objective To investigate the feasibility of gene modification of peripheral blood derived endothelial progenitor cells(EPCs).Methods The rabbit mononuclear cells were collected from rabbit peripheral blood and cultured.The uptaking test of DiI-Ac-LDL was directly visualized with fluorescent microscopy,the immunofluorescence analysis was performed to detect the expression of surface marker.And the human tissue kallikrein(HTK)gene was amplified with PCR,and inserted into pEGFP-N3 vector.EPCs were transfected with the constructed plasmid by means of lipidosome,and the HTK-EGFP fused protein was visualized directly with fluorescent microscopy,and the expression of HTK was detected by RT-PCR and ELISA.Results After 5～6 days of culture,spindle-shaped attached cells clustered together,and cobblestone-like cell layer appeared after 15 days.Cells were positive of uptaking DiI-Ac-LDL and expressing vWF and CD133 related antigen.Plasmids were correctly formed and expressed in the rabbit EPCs.The fused protein had activity of both HTK and EGFP.Conclusion The HTK gene modified EPCs is potential in the treatment of artery injury and endothelial dysfunction.