RESUMO
The current study explored the feasibility and efficacy of co-transfection of the human nerve growth factor (NGF) and vascular endothelial growth factor 165 (VEGF165) genes in rat bone marrow mesenchymal stem cells (BMSCs). The obtained hNGF and vascular endothelial growth factor (VEGF) cDNAs were cloned into the pEGFP-C1 expression vector to construct the recombinant vectors. Co-transfection in rat BMSCs was performed and the expressions of both genes were detected by RT-PCR, Western blot, and enzyme-linked immunospecific assay. The biological activity of recombinant NGF and VEGF proteins was confirmed using the Chick Chorioallantoic Membrane (CAM) assay. NGF and VEGF genes could be expressed successfully in rat BMSCs. The recombinant NGF and VEGF from the rat BMSCs showed a more significant synergetic biological activity compared with single recombinant NGF or VEGF. These findings demonstrate that the co-transfection of hNGF+VEGF genes can enhance the angiogenic effect in vivo.
Assuntos
Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Fator de Crescimento Neural/genética , Transfecção , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Western Blotting , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Embrião de Galinha , Membrana Corioalantoide/metabolismo , Clonagem Molecular , Terapia Genética , Humanos , Fator de Crescimento Neural/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Nerve growth factor (NGF) is required for the differentiation and maintenance of sympathetic and sensory neurons. In the present study, the recombinant expression of human nerve growth factor beta (hNGF-ß) gene in rabbit bone marrow mesenchymal stem cells (rMSCs) was undertaken. Recombinant vector containing hNGF-ß was constructed and transferred into rMSCs, the expressions of the exogenous in rMSCs were determined by reverse transcriptase PCR (RT-PCR), ELISA and Western blot, whereas the biological activity of recombinant hNGF-ß was confirmed using PC12 cells and cultures of dorsal root ganglion neurons from chicken embryos. The results showed that the hNGF-ß gene expressed successfully in the rMSCs, a polypeptide with a molecular weight of 13.2 kDa was detected. The maximal expression level of recombinant hNGF-ß in rMSCs reached 126.8012 pg/10(6) cells, the mean concentration was 96.4473 pg/10(6) cells. The recombinant hNGF-ß in the rMSCs showed full biological activity when compared to commercial recombinant hNGF-ß.
