Isolation and Identification of Human AKT Gene Transfected Human Umbilical Cord Mesenchymal Stem Cells Derived Exosomes

Abstract: In this research， we constructed recombinant adenovirus vector containing human AKT gene (Ad-AKT)， then transfected into human umbilical cord mesenchymal stem cells (hucMSC) and isolated exosomes derived from hucMSC to detect exosomes composition changes which provide experimental basis for the clinical study of exosomes. We designed the primer restriction enzyme with EcoR I， Xho I cleavage site， amplificatied AKT by PCR， and the product was linked into shuttle plasmid labeled by GFP. Linearized recombinant shuttle plasmid was restructured with adenovirus backbone plasmid in Stbl3. The recombinant adenovirus plasmid containing AKT was identified and sequenced by PCR. The recombinant virus plasmid linearized by Pac I endonuclease was transfected into 293A cells to prepare high expression of Ad-AKT， then transfected into hucMSC. The results show that AKT gene recombinant adenovirus is efficiently transfected into hucMSC increasing the expression of AKT protein in hucMSC. AKT protein also increased in the exosomes which derived from hucMSC and the exosomes from AKT transfected hucMSC (AKT-MSC-exosomes). Thus we can obtain exosomes that over expressing target protein by genetic modification， which provides a selective method for the source and the application research of exosomes.

CSTR: 32200.14.cjcb.2014.10.0011