Impact of rAd5-shRNA- PDE5A3 on cGMP in the smooth muscle cells of human corpus cavernosum / 中华男科学杂志

<p><b>OBJECTIVE</b>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 cavernosum, and to provide laboratory evidence for the application of the RNA interference (RNAi) technique for the treatment of erectile dysfunction.</p><p><b>METHODS</b>The recombinant adenovirus rAd5-shRNA-PDE5A3 expressing three pairs of specific shRNA was constructed successfully. The smooth muscle cells of human 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 rAd5-shRNA-PDE5A3 was detected on the cGMP in the smooth muscle cells of the corpus cavernosum.</p><p><b>RESULTS</b>The cGMP level in the smooth muscle cells of the corpus cavernosum was significantly higher in the rAd5-shRNA-PDE5A3 group than in the rAd5-mock control and blank control groups (P < 0.05), most significantly at 72 hours after transfection.</p><p><b>CONCLUSION</b>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.</p>

Impact of adenovirus-mediated PDE5-shRNAs on the free calcium level in the smooth muscle cells of rat corpus cavernosum / 中华男科学杂志

<p><b>OBJECTIVE</b>To observe the influence of recombinant adenovirus-mediated PDE5-shRNAs on the free calcium level in rat penile smooth muscle cells and to explore the feasibility of gene therapy for erectile dysfunction (ED).</p><p><b>METHODS</b>Smooth muscle cells of the rat corpus cavernosum were transfected with constructed rAd-rPDE5-shRNAs and then dyed with the calcium fluorescent probe Fluo-3/AM at 24, 48 and 72 hours. The dynamic changes of the calcium fluorescence intensity were observed under the laser scanning confocal microscope (LSCM). The relative level of intracellular calcium was determined by fluorescence indexes.</p><p><b>RESULTS</b>The fluorescence indexes of calcium at 24, 48 and 72 hours were 829.3 +/- 7.8, 801.5 +/- 9.5 and 856.3 +/- 8.7 in the rAd-rPDES-shRNAs group, significantly lower than in the rAd-mock (1106.3 +/- 10.8, 1121.3 +/- 10.2 and 1058.5 +/- 12.1) and blank control group (1076.6 +/- 9.7, 1133.4 +/- 11.2 and 1104.3 +/- 10.5) (P < 0.05).</p><p><b>CONCLUSION</b>Adenovirus mediated shRNAs of the target PDE5 gene can significantly decrease the intracellular calcium level in the smooth muscle cells of the rat corpus cavernosum.</p>

Differentiation of rat adipose-derived stem cells into smooth-muscle-like cells in vitro / 中华男科学杂志

<p><b>OBJECTIVE</b>To study the feasibility of inducing the differentiation of rat adipose-derived stem cells (ADSCs) into smooth-muscle-like cells in monolayer culture in vitro.</p><p><b>METHODS</b>ADSCs were obtained from the adipose of the inguinal region of the SD rat. A growth curve of the ADSCs was drawn. The fourth passage ADSCs were induced to differentiate into adipocytes with adipogenic inducing fluid and determined by Oil Red O staining, into osteoblasts with osteogenic inducing fluid and determined by Von Kossa staining, as well as into smooth-muscle-like cells with inducing fluid containing beta-mercaptoethanol, and the expression of alpha-smooth muscle actin (alpha-SMA) was detected by the immunohistochemical method.</p><p><b>RESULTS</b>The ADSCs presented spindle and polygon shapes and proliferated rapidly in vitro. The growth curve showed the ADSCs in the logarithmic growth phase two days after subculture. The fourth passage ADSCs exhibited red stained lipid droplets characteristic of adipocytes in the cytoplasm on Oil Red O staining after induction with adipogenic inducing fluid, and calcium nodes on Von Kossa staining after induction with osteogenic inducing fluid. The immunohistochemical results of the ADSCs induced into smooth-muscle-like cells showed that the positive rate of alpha-SMA in the beta-mercaptoethanol induced cells was (29.80 +/- 6.89)%, significantly higher than in the uninduced ones ([2.89 +/- 1.24]%, P < 0.01).</p><p><b>CONCLUSION</b>ADSCs displayed obvious characteristics of smooth muscle cells after induction, and could be a new source of cells in the tissue engineering studies of smooth muscle related diseases.</p>