Lin Guiting, Hayashi Narihiko, Carrion Rafael, Chang Lung-Ji, Lue Tom F, Lin Ching-Shwun
Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California-San Francisco 94143-1695, USA.
J Urol. 2005 Sep;174(3):1142-8. doi: 10.1097/01.ju.0000168615.37949.45.
We investigated whether phosphodiesterase-5 (PDE5) can be down-regulated by a specific small interfering RNA (siRNA) and whether this would improve erectile function.
A PDE5 siRNA encoding oligonucleotide was inserted into pSUPER-retro vector, resulting in the siRNA expressing construct, pPDE5-silencer. The construct was packaged into oncoretroviral particles and then transduced into rat cavernous smooth muscle cells (CSMCs). Cells were examined for PDE5 expression by reverse transcriptase-polymerase chain reaction, Western blotting and immunofluorescence microscopy. Cells were then treated with 10 microM sodium nitroprusside (SNP) and examined for cyclic guanosine monophosphate (cGMP) at 0, 10, 30, 60 and 240 minutes. The siRNA expressing cassette was transferred from the oncoretrovirus to lentivirus, which was then injected into rat penises. Three months later erectile function was examined by electrostimulation and PDE5 expression in cavernous smooth muscle was determined by immunohistochemistry.
CSMCs transfected with pPDE5-silencer (CSMC plus siRNA) showed an 88.2% decrease in PDE5 compared with CSMCs transfected with control vector (CSMCs plus vector). Within 10 minutes of SNP treatment cells (CSMCs, CSMCs plus vector and CSMCs plus siRNA) showed similar sharp increases in cGMP. However, while cGMP levels in CSMCs and CSMCs plus vector returned to almost baseline in 1 hour, the cGMP level in CSMCs plus siRNA remained high even 4 hours after SNP treatment. Rats injected with the siRNA expressing lentivirus showed increased electrostimulated erectile function, as measured by peak intracorporeal pressure and the intracorporeal pressure increase, compared with rats injected with control lentivirus. PDE5 expression was decreased in the siRNA treated cavernous smooth muscle.
PDE5 expression could be decreased by siRNA, resulting in prolonged cGMP accumulation and improved erection.
我们研究了磷酸二酯酶-5（PDE5）是否能被特异性小干扰RNA（siRNA）下调，以及这是否会改善勃起功能。
将编码PDE5 siRNA的寡核苷酸插入pSUPER-retro载体，得到siRNA表达构建体pPDE5-silencer。将该构建体包装成逆转录病毒颗粒，然后转导至大鼠海绵体平滑肌细胞（CSMCs）。通过逆转录聚合酶链反应、蛋白质印迹法和免疫荧光显微镜检查细胞中的PDE5表达。然后用10微摩尔硝普钠（SNP）处理细胞，并在0、10、30、60和240分钟时检测环磷酸鸟苷（cGMP）。将siRNA表达盒从逆转录病毒转移至慢病毒，然后将慢病毒注射到大鼠阴茎中。三个月后，通过电刺激检查勃起功能，并通过免疫组织化学法测定海绵体平滑肌中的PDE5表达。
与转染对照载体的CSMCs（CSMCs加载体）相比，用pPDE5-silencer转染的CSMCs（CSMC加siRNA）中PDE5减少了88.2%。在SNP处理细胞（CSMCs、CSMC加载体和CSMC加siRNA）的10分钟内，cGMP呈现相似的急剧增加。然而，虽然CSMCs和CSMC加载体中的cGMP水平在1小时内几乎恢复到基线，但即使在SNP处理4小时后，CSMC加siRNA中的cGMP水平仍保持较高。与注射对照慢病毒的大鼠相比，注射表达siRNA慢病毒的大鼠经电刺激后的勃起功能增强，通过海绵体内峰值压力和海绵体内压力增加来衡量。在经siRNA处理的海绵体平滑肌中，PDE5表达降低。
siRNA可降低PDE5表达，导致cGMP积累延长并改善勃起。
