Lentiviral vector mediated CGRP gene in vitro transfection and its effects on biological properties of MSC / 重庆医学

ABSTRACT

Objective To explore in vitro mesenchymal stem cell (MSC) transfection of lentiviral vector mediated calcitonin gene‐related peptide(CGRP) gene and its effects on biological properties of MSC .Methods MSC were isolated ,cultured and identi‐fied .MSC were infected by lentivirus encoding recombinant enhanced green fluorescent protein (EGFP) gene and CGRP (Lv‐EG‐FP‐CGRP) .The transfection efficiency was determined by the inverted fluorescence microscope and flow cytometry .The expression levels of CGRP were detected in CGRP‐modified MSC by using real‐time PCR ,immunocytochemistry and enzyme‐linked immu‐nosorbent assay (ELISA) .The proliferation ,aging and differentiation ability of MSC were evaluated by MTT ,β‐galactosidase stai‐ning and inducing differentiation respectively .Results After 48 h of MSC transfection by Lv‐EGFP‐CGRP ,EGFP/CGRP could be expressed stably .When multiplicity of infection (MOI) was 30 ,the transfection efficiency reached more than 80% .Compared with the MSC group and the MSC‐EGFP group ,the mRNA and protein expression levels of CGRP in CGRP‐modified MSC(MSC‐CGRP group) were markedly increased(all P<0 .01) .The results of MTT ,β‐galactosidase staining and inducing differentiation assay dem‐onstrated that the transfected CGRP basically had no effect on the proliferation ,aging and endotheliocyte differerntiation of MSC . Conclusion MSC is a kind of ideal genetic vector cell ,which can serve as the target cell of CGRP gene transduction for the applica‐tion of gene therapy and lays the foundation for follow‐up in vitro and vivo experiments .