Effect of phosphodiesterase 5 inhibition by lentivirus-mediated stable gene silencing on survival of adipose-derived stem cells

Abstract

Objective To explore the in vitro effect of lentiviral expressions vector carrying phosphodiesterase 5 (PDE5)-shRNA gene on proliferation, apoptosis and secretion of adipose-derived stem cells (ADSCs). Methods (1) ADSCs were separated and purified from the rats, sub-cultivation and identification were performed. Lentiviral expression vectors carried different PDE5 shRNA genes (PDE5 shRNA1, PDE5 shRNA2 and PDE5 shRNA3) were established and negative controls (NC shRNA) were used. The above vectors were transfected into ADSCs with lentivirus package; Western blotting was carried out to detect the PDE5 protein expression level and real time-PCR was carried out to detect the mRNA expression level so as to select the most efficient cell line. (2) ADSCs were divided into four groups: normal control group, ischemia-re-oxygenation (I/R) injury group (normal cells+ I/R injury), NC group (NC shRNA-ADSCs+I/R injury) and experimental group (PDE5 shRNA-ADSCs+I/R injury); CCK-8 assay and Edu method were performed to evaluate the proliferation of cells, flow cytometry was employed to detect the apoptosis of ADSCs, and ELISA was used to quantify the protein expressions of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (b-FGF) and cyclic guanosine monophosphate (cGMP). Results Lentiviral expression vectors carried different PDE5 shRNA genes were successfully established and PDE5 sh ADSCs with stable PDE5 expression were established. As compared with cells from normal control group, cells from I/R injury group had significantly decreased ADSCs proliferation rate and increased apoptosis rate at early and late stages, and significantly increased VEGF and FGF levels in the liquid supernatant and cGMP level in the cells (P 0.05); as compared with the NC group, the experimental group had significantly increased ADSCs proliferation rate and decreased apoptosis rate, and significantly increased VEGF and FGF levels in the liquid supernatant and cGMP level in the cells (P<0.05). Conclusion The proliferation, apoptosis and secretion of ADSCs could be effectively improved and the stem cell efficiency was modified via upregulating cGMP level after PDE5 gene expression being inhibited in ADSCs. Key words: Adipose-derived stem cell; Phoshodiesterase 5; Ischemia/re-oxygenation; Proliferation; Apoptosis