靶向人PDESA3基因的shRNA对人阴茎海绵体平滑肌细胞cGMP的影响

目的：观察腺病毒载体介导特异性短发夹RNA（shRNA）对人阴茎海绵体平滑肌细胞环磷酸鸟苷（cGMP）的影响，为应用RNA干扰（RNAi）技术治疗阴茎勃起功能障碍（ED）提供实验依据。方法：成功构建携带3条针对人PDESA3基因位点特异性shRNA的重组腺病毒（rAd5-shRNA-PDE5A3），并设立阴性对照病毒组和空白对照组，分别转染人阴茎海绵体平滑肌细胞。以放射免疫法分别检测转染重组腺病毒24、48、72h后细胞内cGMP浓度变化，观察rAd5-shRNA-PDE5A3对海绵体平滑肌细胞内cGMP的影响。结果：实验组rA（15-shRNA-PDE5A3转染人阴茎海绵体平滑肌细胞后胞内cGMP水平显著高于阴性对照组和空白对照组，在转染后72h最为显著。结论：3条特异性针对人PDE5A3mRNA靶位点的shRNA可有效地增加海绵体平滑肌细胞内cGMP的水平，增强对PDE5基因的阻抑效果。

Impact of rAdS-shRNA- PDE5A3 on cGMP in the Smooth Muscle Cells of Human Corpus Cavernosum

Abstract： Objective： To investigate the effect of phosphodiesterase type 5 （PDE5） small interfering RNA （siRNA） on cyclic guanosine monophosphatethe （cGMP） in the smooth muscle cells of human corpus cavemosum, and to provide laboratory evidence for the application of the RNA interference （RNAi） technique for the treatment of erectile dysfunction. Methods ： The recombinant adenovirus rAdS-shRNA-PDESA3 expressing three pairs of specific shRNA was constructed successfully. The smooth muscle cells of hu- man corpus cavernosum were divided into an experimental, a negative control and a blank control group, and transfected respectively with rAd5-shRNA-PDE5A3, adenovirus rAd5-mock and phosphate buffered saline. The concentration of cGMP was measured by radioimmunoassay at 24, 48 and 72 hours after transfection, and the effect of rAdS-shRNA-PDE5A3 was detected on the cGMP in the smooth muscle cells of the corpus cavernosum. Results ： The cGMP level in the smooth muscle cells of the corpus cavemosum was significantly higher in the rAd5-shRNA-PDESA3 group than in the fAdS-mock control and blank control groups （ P 〈 0.05 ）, most significantly at 72 hours after transfection. Conclusion： The rAd5-shRNA-PDE5A3 can obviously increase the cGMP level in the smooth muscle cells of human corpus cavernosum, and enhance the inhibition of the PDE5 gene.