PDE5基因siRNA对人阴茎海绵体平滑肌细胞cGMP的影响

目的:观察PDE5基因小干扰RNA(siRNA)对人阴茎海绵体平滑肌细胞环磷酸鸟苷(cGMP)的影响,为阴茎勃起功能障碍(ED)的基因治疗提供实验依据。方法:使用美国Amb ion公司提供的设计软件设计并合成人PDE5基因的siRNA序列,合成3对PDE5 siRNA和1对阴性对照siRNA,转染人阴茎海绵体平滑肌细胞,同时设定空白转染组为对照组。以酶联免疫法分别检测转染后不同时间(24、48、72、96 h)点海绵体平滑肌细胞内cGMP浓度变化,观察PDE5 siRNA对海绵体平滑肌细胞内cGMP的影响。结果:siRNA1、siRNA2和siRNA3转染后人阴茎海绵体平滑肌细胞cGMP水平显著高于阴性对照siRNA组和空白对照组(P<0.05),在转染后72 h最为显著,siRNA1、siRNA2、siRNA3、阴性对照siRNA和空白对照组cGMP测定值分别为:5.89±0.19、3.52±0.16、2.88±0.08、0.72±0.12、0.60±0.16 pmol/m l,siRNA1组明显高于siRNA2、siRNA3组(P<0.05)。结论:体外化学合成的PDE5 siRNA能有效地增加海绵体平滑肌细胞内cGMP的水平,不同序列siRNA增加cGMP水平的能力不同,为ED的基因治疗提供了新思路。

Phosphodiesterase Type 5 siRNA Increases cGMP in the Smooth Muscle Cells of Human Corpus Cavernosum

Objective:To investigate the effect of phosphodiesterase type 5(PDE5) small interfering RNA(siRNA) on the cGMP in the smooth muscle cells of human corpus cavernosum,and to provide an experimental groundwork for the gene therapy of erectile dysfunction(ED).Methods: Small interfering RNAs targeting PDE5 gene were synthesized by using web design software provided by Ambion,three siRNAs and a control siRNA were synthesized by Ambion.siRNAs were transfected into the smooth muscle cells of human corpus cavernosum by using siPORT~(TM) Lipid reagent.cGMP was detected by ELISA at different times(24,48,72 and 96 h) after transfection.Results: The cGMP levels of the siRNA1,siRNA2 and siRNA3 groups were significantly higher than those of the siRNA control and blank control groups(P<(0.05)),and so was it in the siRNA1 group than the siRNA2 and siRNA3 groups (P<(0.05)),with significant difference between the siRNA control and the blank control group(P>(0.05)).Conclusion: The synthesized siRNAs in vitro are capable of increasing the level of cGMP in the smooth muscle cells of human corpus cavernosum,different siRNAs with different capabilities.The siRNA technique could provide not only an extremely powerful tool for the functional analysis of genome but also a new approach to ED gene therapy.