AB184. Effects of target gene expression on ex vivo differentiated mesenchymal stromal cells transfected by lent viral vector with PDE5 of short hairpin RNA

Abstract

IntroductionIt has been shown that the major causes of erectile dysfunction (ED) associated with aging were via a diffuse and progressive corporal fibrosis to cavernous smooth muscle cells (CSMCs) in the penis. Previous studies have demonstrated that bone marrow-mesenchymal stromal cells (BM-MSCs) could possess the ability for self-renewal, multiline age differentiation and in vivo functional regeneration of damaged tissues. It was suggested that they be induced to differentiate into smooth muscle-like cells in vitro and have a potential source for stem-cell-based gene therapy of ED. Specific phosphodiesterases (PDE5) has been proved to play an important role in regulating penile erection to return to flaccidity by hydrolysis of cyclic guanosine monophosphate (cGMP) as a specific hydratase. Therefore, inhibiting specifically the catalytic activity of the target gene PDE5 has been indicated to promote the accumulation of cGMP and enhance erection. In recent years, the introduction of short hairpin RNA (shRNA) has proved to be a powerful tool for suppressing strongly gene specific expression through a process known as RNA interference. This study was to describe that stem cell-based replacement therapy with rat BM-MSCs by transfected the lentiviral vector carrying silencing target gene PDE5 of short hairpin RNA (PDE5-shRNA) could aid in the regeneration of penile smooth musculature and might help to benefit recovery of erectile function.PurposeTo investigate the expression of PDE5 in ex vivo differentiation of gene-modified BM-MSCs using the lentiviral vector containing silencing target gene PDE5 of shRNA.Methods and materialsSD rat bone marrow-derived MSCs were separated, cultured and purified in vitro by Percoll density gradient centrifugation combined with adherent culture. The third passage rat BM-MSCs were obtained, and were identified by cell surface markers with flow cytometry (FCM). The lentiviral vector carrying silencing target gene PDE5 of shRNA was constructed and then transfected into the third passage rat BM-MSCs. The gene-modified rat BM-MSCs were induced to differentiate into smooth muscle-like cells exposed to VEGF and b-FGF median vitro. The proliferative ability of these cells was tested by cell counting kit 8 (CCK-8). Smooth muscle-like cells differentiation was assessed by immunofluorescence and then subjected to immunocytochemistry for specific markers of α-smooth muscle actin (α-SMA). Real-time quantitative PCR, immunohistochemical staining and Western blot analysis for PDE5 gene expression in differentiated smooth muscle-like cells were carried out.ResultsAfter the conducted lentiviral vector containing PDE5-shRNA was transfected into rat BM-MSCs, the expression of EGFP was detected at 24 hands it became the strongest at 72 h, which FCM showed the transfection efficiency were 91.3% at this time. The EGFP expression rates of rat BM-MSCs transfected with PDE5-shRNA at 3 d, 7 d, 14 d after transduction were 91.3%, 86.1%, and 82.7%, respectively. There was still visible green fluorescence at 28 d after transfection. The gene-modified rat BM-MSCs with PDE5-shRNA were successfully induced to differentiate into smooth muscle-like cells. Transduction of the lentiviral vector PDE5-shRNA into rat BM-MSCs led to down-regulation of PDE5. The expression of PDE5 was reduced 67.2% by PDE5-shRNA compared with the control lentiviral vector. The proliferative property of rat BM-MSCs did not significantly change among those transfected with PDE5-shRNA (P<0.001). The differentiated rat BM-MSCs were identified to smooth muscle-like cells by immunocytochemistry.ConclusionsThese results suggest that the lentiviral vector with gene-specific silencing PDE5 of shRNA could effectively transfect into rat BM-MSCs. They reveal that the gene-modified rat BM-MSCs with PDE5-shRNAcould be successfully induced to differentiate into smooth muscle-like cells in vitro and inhibit the expression of target gene PDE5.SupportThis work was supported by National Natural Science Foundation of China (81050026, 81370702).