O 18. Mesenchymal stromal cells transfected by recombinant adenovirus vector with short hairpin RNA of silencing PDE5

O 18. Mesenchymal stromal cells transfected by recombinant adenovirus vector with short hairpin RNA of silencing PDE5

Heng-Jun Xiao1, MinXia2, Jun Chen3, Xiao-Peng Liu1, Hao Zhang1, YanZhang1, Jian-Guang Qiu1, Xin Gao1

1Department of Urology, the Third Affiliated Hospital of Sun Yat-sen University, China; 2School of public health, Sun Yat-sen University, China; 3Department of infertility&sexual medicine, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, Guangdong Province, China

Introduction: Many studies indicate that bone marrow me s enchymal stromal cells (MSCs) not only have multidifferentiation potential and functional regeneration of impaired tissues, but also are relatively easy to isolate and expand ex vivo. In view of these properties, they can be a useful vehicle for gene delivery in adult stem cell-based gene therapy. Specific phosphodiesterases (PDE5) has been shown to regulate penile erection to return to flaccidity by hydrolysis of cyclic guanosine monophosphate (cGMP). 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 the transfection efficiency of the recombinant adenovirus vector with gene-specific silencing PDE5 of short hairpin RNA transducing into rat MSCs.

Objective: To construct the recombinant adenovirus vectors with short hairpin RNA of silencing PDE5 and investigate the adenoviral transfection efficiency by transducing into rMSCs.

Methods: The recombinant adenovirus vector carrying shRNA with inhibiting the specific expression of PDE5 (pAd-PDE5- shRNA) and enhanced green fluorescent protein (EGFP) gene was constructed. 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 rMSCs were obtained, and were identified by cell surface markers with flow cytometry (FCM). The EGFP gene expression was verified by the intensity of green fluorescence under fluorescence microscope and the transfection efficiency was assessed by FCM in ex vivo expanded rMSCs transfected with pAd-PDE5-shRNA.

Results: The recombinant adenovirus vector pAd-PDE5-shRNA was successfully constructed. After pAd-PDE5-shRNA was transfected into rMSCs, the expression of EGFP was detected at 24h and it became the strongest at 72 h, which FCM showed the transfection efficiency were 88.6% at this time. The EGFP expression rates of rMSCs transfected with pAd-PDE5-shRNA at 3 d, 7 d, and 14 d after transduction were 88.6%, 85.2%, and 78.5%, respectively. There was still visible green fluorescence at 28d after transfection.

Conclusions: The recombinant adenovirus vector with genespecific silencing PDE5 of shRNA can effectively transfect into rMSCs in vitro. Gene-modified rMSCs with pAd-PDE5-shRNA may have a potential cell source of stem-cell-based therapy for ED.

Key words

Mesenchymal stromal cells; recombinant adenovirus vector; short hairpin RNA of silencing PDE5

DOI: 10.3978/j.issn.2223-4683.2012.s143

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