AB115. Effects of target gene expression on ex vivo differentiated mesenchymal stromal cells transfected by lentiviral vector with PDE5 of short hairpin RNA

Objective: To 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: SD 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 media in 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.

Results: After the conducted lentiviral vector containing PDE5-shRNA was transfected into rat BM-MSCs, the expression of EGFP was detected at 24 h and it became the strongest at 72 h, which FCM showed the transfection efficiency was 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.

Conclusions: These results suggest that the lentiviral vector with gene-specific silencing PDE5 of shRNA could effectively transfect #I# rat BM-MSCs. They reveal that the gene-modified rat BM-MSCs with PDE5-shRNA could be successfully induced to differentiate into smooth muscle-like cells in vitro and inhibit the expression of target gene PDE5.

Keywords: Erectile dysfunction; marrow-mesenchymal stromal cells; phosphodiesterases 5