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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PDE5A3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rAd5-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。
OBJECTIVE: To investigate the effect of adenovirus-mediated short hairpin RNA (shRNA) on cyclic guanosine monophosphate (cGMP) in human corpus cavernosum smooth muscle cells. To study the effect of RNA interference (RNAi) on erectile dysfunction (ED) Provide experimental basis. Methods: Three recombinant adenovirus (rAd5-shRNA-PDE5A3) carrying human PDE5A3 gene-specific shRNA were constructed and transfected into human corpus cavernosum smooth muscle cells by negative control group and blank control group respectively. The concentration of cGMP in transfected cells was detected by radioimmunoassay at 24, 48 and 72 hours, and the effect of rAd5-shRNA-PDE5A3 on cGMP in smooth muscle cells was observed. Results: The intracellular cGMP level of rAd5-shRNA-PDE5A3 transfected human corpus cavernosal smooth muscle cells was significantly higher than that of negative control group and blank control group, which was the most significant at 72h after transfection. CONCLUSIONS: Three shRNAs specific to human PDE5A3 mRNA target site can effectively increase the level of cGMP in SMC and increase the inhibitory effect on PDE5 gene.