短发夹RNA对大鼠阴茎海绵体平滑肌细胞磷酸二酯酶5型基因表达的抑制作用

目的 观察短发夹RNA(shRNA)对大鼠阴茎海绵体平滑肌细胞磷酸二酯酶5型(PDE5)基因表达的抑制作用,探讨运用RNA干扰(RNAi)技术治疗勃起功能障碍(ED)的可行性.方法 构建靶向大鼠PDE5基因的shRNA重组腺病毒rAd-rPDE5-shRNA,将其转染大鼠阴茎海绵体平滑肌细胞48 h后,通过荧光标签进行显微计数确定转染效率,并以逆转录-聚合酶链反应(RT-PCR)及Western blot检测PDE5基因的表达水平.结果 rAd-rPDE5-shRNA构建成功,转染大鼠阴茎海绵体平滑肌细胞效率达95%以上,并使PDE5基因表达在mRNA水平抑制(80.78±2.30)%,在蛋白水平抑制(67.39±3.33)%.结论 以腺病毒为载体表达的shRNA能稳定、有效地抑制大鼠阴茎海绵体平滑肌细胞PDE5基因的表达.

The inhibitive effect of shRNA on the phosphodiesterase type 5 in smooth muscle cells of rat corpus cavernosum

Objective To down-regulate the expression of phosphodiesterase type 5 (PDE5) gene by short hairpin RNA (shRNA) in smooth muscle cells of rat corpus cavernosum,and investigate the feasibility of gene therapy for erectile dysfunction (ED). Methods A recombinant adenovirus, rAd-rP-DE5-shRNA, targeting the PDE5 gene of rats was constructed. The rAd-rPDE5-shRNA was transfected into the smooth muscle cells of rat corpus cavernosum. The efficiency of transfection was evaluated by cell counting of EGFP expressed cells under fluorescence microscopy, and the expression of PDE5 gene was de-tected by RT-PCR and Western blot at the 48th h post-transfection. Results rAd-rPDES-shRNA was i-dentified and transfected at a MOI of 30 pfu per call for 48 h. The expression of PDE5 in the smooth mus-cle cells of rat corpus cavernosum was inhibited by (80.78±2.30)% at mRNA level, and (67.39± 3.33)% at protein level. Conclusion The shRNA delivered by the recombinant adenovirus can stably and effectively inhibit the expression of PDE5 in smooth muscle cells of rat corpus cavernosum.