高血压大鼠海绵体平滑肌细胞间连接的变化

目的:比较自发性高血压大鼠(SHR)和正常血压大鼠阴茎海绵体平滑肌细胞间连接改变及与阴茎勃起功能的关系。方法:注射阿朴吗啡(APO)观察14周龄SHR(SHR组,n=5)、W istar-Kyoto大鼠(WKY组,n=5)阴茎勃起情况,用透射电镜观察其阴茎海绵体平滑肌细胞间连接超微结构,RT-PCR测定海绵体平滑肌细胞Connexin 43的mRNA表达,免疫组化观察Connexin 43蛋白表达。结果:SHR组大鼠阴茎勃起次数明显低于WKY组(P<0.05),电镜发现SHR组大鼠阴茎海绵体平滑肌细胞间大量胶原纤维增生,Connexin 43蛋白及其mRNA表达较WKY组显著降低(P<0.05)。结论:高血压影响阴茎勃起功能,阴茎海绵体平滑肌细胞间连接的病理改变可能是高血压性勃起功能障碍的发病机制之一。     

干细胞治疗阴茎勃起功能障碍的研究进展

阴茎勃起功能障碍(ED)是指男性反复或者持续性的难以达到和维持充分的阴茎勃起,无法完成性交或满意性活动的病理现象。海绵体神经(CN)损伤引起的勃起神经反射中断,是患者出现ED的直接原因,此外,CN损伤后,阴茎海绵体组织平滑肌细胞和内皮细胞凋亡增加,海绵体平滑肌纤维数量减少加重了ED的发生。因此,尽早干预CN损伤的病理过程,促进CN再生是治疗CN损伤性ED的关键。近年来,干细胞在ED治疗中的应用日益成为临床研究热点。现对胚胎干细胞(ESC)、间充质干细胞(MSCs)、肌源性干细胞(MDSCs)、脂肪干细胞(ADSCs)在ED治疗中的研究综述如下。     

The TGF-β1/Smad/CTGF pathway and corpus cavernosum fibrous-muscular alterations in rats with streptozotocin-induced diabetes

Diabetes-associated erectile dysfunction is associated with increased extracellular matrix deposition and reduced smooth muscle content in the corpus cavernosum. The mechanisms of these processes are not well understood. In this study, we investigated fibromuscular changes in the corpus cavernosum of rats with streptozotocin-induced diabetes to determine the mechanisms underlying pathologic changes in penile structure and function. Forty 8-week-old Sprague-Dawley rats were randomly distributed into control and diabetic groups. Diabetes was induced by a one-time intraperitoneal injection of streptozotocin 60 mg/kg. Twelve weeks later, erectile function was measured by cavernous nerve electrostimulation with real-time intracorporal pressure assessment. The penis was harvested for histologic examination (Masson trichrome stain, picrosirius red stain, Hart elastin stain, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, and immunohistochemistry) and Western blot. Diabetes significantly attenuated erectile response to cavernous nerve electrostimulation. Diabetic animals exhibited a decreased smooth muscle/collagen ratio in the corpus cavernosum. The ratio of collagen I to II fibers was significantly lower in the corpora of diabetic rats compared with controls. Cavernous elastic fibers were fragmented in diabetic rats. There was up-regulation of the transforming growth factor β1/Smad/connective tissue growth factor signaling pathway in diabetic rats. Phospho-Smad2 expression was higher in smooth muscle cells and fibroblasts of diabetic rats, as was the apoptotic index. The up-regulation of the transforming growth factor β1/Smad/connective tissue growth factor signaling pathway might play an important role in diabetes-induced fibrous-muscular structural changes and deterioration of erectile function.     

Organization and relative content of smooth muscle cells, collagen and elastic fibers in the corpus cavernosum of rat penis

PURPOSE: The corpus cavernosum smooth muscle and extracellular matrix are essential for normal penile erection and are implicated in erectile dysfunction. Although investigations of these issues have used the rat corpus cavernosum, organization of its components is to date not well known. We characterized and quantified the smooth muscle cells and the main extracellular matrix components of the rat corpus cavernosum. MATERIALS AND METHODS: Collagen, elastic fibers and smooth muscle cells were stained on paraffin sections of rat penises using sirius red and Gomori's reticulin, Weigert's resorcin-fuchsin and an anti-smooth muscle cells alpha-actin antibody, respectively. Stained components were then quantified by computer aided morphometry. RESULTS: Smooth muscle cells were restricted to the subendothelial space of corpus cavernosum and had a volumetric density of 9.1%. Collagen was thick, usually in transversely oriented bundles and was the most abundant component of the trabeculae with a volumetric density of 62.7%. Gomori's reticulin disclosed a meshwork of fibrils also in the subendothelial space but did not stain the thicker bundles. Volumetric density of elastic fibers was 4.9%, and at the periphery of the corpus cavernosum the fibers were parallel to the long axis of the penis, while in deeper regions most of them were transversely oriented and at different directions from those of collagen. CONCLUSIONS: Rat corpus cavernosum differs from that of humans by lesser amounts of smooth muscle cells, greater amounts of collagen and the presence of fibrillar collagen and smooth muscle cell subendothelial layers. Therefore, these differences should be considered when using the rat penis for studies on erection.     

小干扰RNA抑制人阴茎海绵体平滑肌细胞间隙连接蛋白connexin43的表达

目的:利用小干扰RNA(siRNA)抑制人阴茎海绵体平滑肌细胞间隙连接蛋白connexin43(Cx43)的表达和检测细胞间间隙连接通讯功能,探讨该技术在阴茎海绵体平滑肌细胞间隙连接和阴茎勃起功能研究中的应用。方法:利用Ambion公司设计软件,构建靶向人Cx43基因的siRNA重组质粒,转染人阴茎海绵体平滑肌细胞48h后,逆转录-聚合酶链反应(RT-PCR)和Western印迹检测Cx43基因和蛋白的相对表达水平、划痕标记荧光染料传输技术检测细胞间间隙通讯功能,并分别与siRNA阴性对照、空白对照组比较。结果:酶切和测序证实siRNA真核表达载体构建成功。siRNA重组质粒转染细胞后的Cx43 mRNA和蛋白相对表达水平分别为(0.45±0.08)%、(0.56±0.06)%,与siRNA阴性对照组(0.72±0.04)%、(0.80±0.08)%和空白对照组(0.74±0.09)%、(0.77±0.11)%相比,差异均有显著性(P(0.05);转染后的细胞间间隙连接通讯功能也显著降低。结论:siRNA能有效抑制人阴茎海绵体平滑肌细胞Cx43的表达和阻断间隙连接介导的间隙连接通讯功能。     

衰老对大鼠阴茎勃起机制的影响

为探讨衰老对阴茎勃起机制的影响程度,以不同月龄的雄性大鼠分别观察海绵体神经诱导的阴茎勃起,海绵体平滑肌的舒张功能以及阴茎含ＮＯＳ神经和平滑肌量。结果表明电刺激老年鼠海绵体神经可取得与年轻和中年鼠相当的勃起压力,但勃起潜伏期较长。注射罂粟碱提示老年海绵体平滑肌顺应性较年轻鼠差。检查还提示老年鼠阴茎内含ＮＯＳ神经和平滑肌纤维量有一定减少。实验证实衰老对阴茎勃起机制中关键性的组织结果和功能有一定影响,但     

Vascular impotence: focal or diffuse penile disease.

Using computerized image analysis the percentage of smooth muscle fibers was measured in several biopsies of the penis from 10 cadavers and 5 patients with vascular impotence. No significant difference was observed between the proximal and distal areas, and/or between the peripheral and central areas in 1 corpus cavernosum, and between the 2 corpora cavernosa for each patient or cadaver. The percentage of smooth muscle fibers was less in impotent patients in comparison with the cadavers, in which the erectile status was not known, and this reduction occurred throughout both cavernous bodies. Vascular impotence is a diffuse penile disease. Therefore, a cavernous body biopsy can be used to study the penile structure in the assessment of vascular impotence.     

Human neural crest stem cells transplanted in rat penile corpus cavernosum to repair erectile dysfunction

OBJECTIVE

To investigate the feasibility of applying neural crest stem cells (NCSCs), with multipotent capacity, to repair injury in the penile cavernosum, the HNC10.K10 (K10) immortalized NCSC line was transplanted into the penile cavernosum of adult rats, as one of the causes of erectile dysfunction is damaged penile cavernous smooth muscle cells and sinus endothelial cells.

MATERIALS AND METHODS

The K10 human NCSC line was generated via transfection of primary cultured NCSC with a retroviral vector encoding v‐myc. K10 NCSCs were transplanted into the cavernosum of adult rats. The expression of cell type‐specific markers for endothelial cells (CD31 and von Willebrand factor), and specific markers for smooth muscle cells (smooth muscle cell actin, calponin, and desmin) was determined immunohistochemically in the penile cavernosum of rats 2 weeks after transplantation.

RESULTS

In the rat cavernosum, transplanted K10 NCSCs identified by human nuclear antigen labelling expressed cell type‐specific markers for endothelial cells (CD31 and von Willebrand factor), and specific markers for smooth muscle cells (smooth muscle cell actin, calponin, and desmin) 2 weeks after transplantation. Human NCSCs transplanted into the rat penile corpus cavernosum differentiated into endothelial cells or smooth muscle cells, as shown by their expression of cell type‐specific markers for the cell types.

CONCLUSION

It appears that NCSCs are an ideal cell source for reconstructing endothelial and smooth muscle cells in the corpus cavernosum in cell therapy for patients with erectile dysfunction.     

Biochemical changes in cavernosal tissue caused by single sided cavernosal nerve resection and the effects of alpha lipoic acid on these changes

ObjectiveOne of the most important complications of radical prostatectomy operation is erectile disfunction (ED). Oxidative stress patterns and apoptotic changes may happen in smooth muscles and endothelial cells of corpus cavernosum after neuropraxia or neurectomy. Alpha lipoic acid (ALA) shows its antioxidant properties by eliminating free radicals. In this experimental study we investigated the effects of ALA on rehabilitation of cavernosal tissue and nitric oxide synthase (NOS) containing nerve fibers on erectile tissue.Materials and methodsIn this study four groups were formed by inclusion of 63 adult fertile rats. Control group (n: 9), sham operation group (n: 18), 18 rats underwent unilateral neurectomy of a 5-mm. segment of the cavernous nerve (group DI) and another 18 rats group which ALA received after unilateral neurectomy (group DII). Assessments were done 3 weeks after neurectomy.ResultsWe assessed number of NOS containing nerve fibers via nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase staining. According to NADPH diaphorase staining group DII significantly recovered comparing group DI (48.89±19.00 and 17.22±6.67 respectively) (p<0.05). SOD activity is reduced in both; group DI and group DII (31.42±6.06 and 40.38±4.24). Nitrite+nitrate levels were elevated significantly in both group DI and group DII (0.52±0.05 and 0.44±0.02 micromole/gr wet tissue respectively) when compared with other groups (p<0.05). There is no statistical difference between results of group DI and Group DII (p>0.05).ConclusionThis study confirms that neurectomy caused decrease of intracavernous pressure and number of NOS fibers. Neurectomy and surgical trauma caused oxidative stress in rat corpus cavernosum. As a potent antioxidant ALA has positive effects on cavernosal tissue regeneration and rehabilitation by reducing oxidative stress. In this aspect, ALA may have a potential advantage in penile rehabilitation after radical prostatectomy.     

Effects of Icariside II on Improving Erectile Function in Rats With Streptozotocin-Induced Diabetes

Icariin and icariside II (ICA II), 2 active components isolated from herba epimedii, have a closely structural relationship. There is evidence that icariin may be useful in the treatment of erectile dysfunction (ED); however, the study on the therapeutic efficacy of ICA II on ED is currently scant. We investigated the effects of ICA II on improving erectile function of rats with streptozocin-induced diabetes. Fifty 8-week-old Sprague-Dawley rats were randomly distributed into normal control and diabetic groups. Diabetes was induced by a one-time intraperitoneal injection of streptozocin (60 mg/kg). Three days later, the diabetic rats were randomly divided into 4 groups including a saline-treated placebo arm and 3 ICA II-treated models (1, 5, and 10 mg/kg/d). After 3 months, penile hemodynamics was measured by cavernous nerve electrostimulation (CNE) with real time intracorporal pressure assessment. Penises were harvested with subsequent histological examination (picrosirius red stain, Hart elastin stain, and immunohistochemical stain) and Western blots to explore the expression of the nitric oxide-cyclic guanosine monophosphate (NO-cGMP) and transforming growth factor β1 (TGFβ1)/Smad2 signaling pathways. Diabetes significantly attenuated erectile responses to CNE. Diabetic rats had decreased corpus cavernosum smooth muscle/collagen ratio and endothelial cell content relative to the control group. The ratio of collagen I to III was significantly lower in the corpora of diabetic rats; furthermore, cavernous elastic fibers were fragmented in the diabetic animals. Neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase, and vascular endothelial growth factor were expressed at lower levels in the diabetic group; ICA II-treated diabetic rats had higher expression in the penis relative to placebo-treated diabetic animals. Both the TGFβ1/Smad2/connective tissue growth factor (CTGF) signaling pathway and apoptosis were down-regulated in the penis from ICA II-treated rats. ICA II treatment attenuates diabetes-related impairment of penile hemodynamics, likely by increasing smooth muscle, endothelial function, and nNOS expression. ICA II could alter corpus cavernosum fibrous-muscular pathological structure in diabetic rats, which could be regulated by the TGFβ1/Smad2/CTGF and NO-cGMP signaling pathways.     

内源性一氧化碳对离体犬阴茎海绵体平滑肌的作用

目的:探讨内源性一氧化碳(CO)对离体犬阴茎海绵体平滑肌作用的影响。方法:利用水浴条件下阴茎海绵体肌条的张力测定技术,用CO合成的关键酶血红素氧合酶(HO)的诱导剂———氯高铁血红素诱导海绵体平滑肌生成内源性CO,观察CO对去氧肾上腺素(PE)诱导收缩的阴茎海绵体肌条作用的影响。结果:氯高铁血红素对10μmol/L PE诱导的肌条收缩具有浓度依赖性的松弛作用,10~100μmol/L氯高铁血红素对平滑肌肌条的松弛效应与空白对照相比明显升高(P<0.01)。用锌原卟啉-Ⅸ(ZnPP-Ⅸ)或亚甲蓝孵育处理后的肌条,氯高铁血红素的舒张作用明显减弱(P<0.01)。结论:内源性CO具有浓度依赖性松弛阴茎海绵体平滑肌的作用,其机制可能是通过CO环磷酸鸟苷途径作用所致。     

豚鼠阴茎海绵体中ICC和NO信号通路的形态学关系

目的初步探讨豚鼠阴茎海绵体组织中cajal间质细胞（ICC）和神经型一氧化氮合成酶（nNOS）阳性神经元的分布及两者的组织学关系。方法①制作成年豚鼠阴茎海绵体组织冰冻切片,利用c-kit/nNOS免疫荧光双染技术,激光共聚焦扫描显微镜（LSCM）下观察阴茎海绵体ICC及nNOS阳性神经元的形态及分布情况。②酶消化法体外培养阴茎海绵体ICC,c-kit/nNOS免疫荧光双染,LSCM下观察细胞形态及c-kit和nNOS的表达情况。结果 ICC分布在阴茎海绵体平滑肌小梁边缘及平滑肌肌间,纺锤形,具有长的突起,胞体呈圆形或椭圆形,并且表达nNOS;阴茎海绵体平滑肌小梁边缘及平滑肌肌间同时分布有ICC和nNOS阳性神经元,局部nNOS阳性神经主干附近可见较多ICC分布。结论豚鼠阴茎海绵体ICC与nNOS阳性神经元组织联系紧密,ICC可能参与阴茎海绵体内NO神经信号的传递。     

Role of penile electrodermal activity in the evaluation of autonomic innervation of corpus cavernosum

Electrodiagnostic tests measuring the activities of cavernous smooth muscle and sudomotor structures of penile skin are used in order to evaluate autonomic innervation of the penis. Owing to closeness of these tissues, the interference of sympathetic activity during recording is a possibility. In this study, we investigated this possibility in 10 patients whose cavernosal tissues were destroyed during penile prosthesis implantation by comparing the pre- and postoperative penile skin electrodermal activities. Penile electrodermal activities were recorded with surface electrodes before and after the operation. All of the patients had spontaneous and evoked penile electrodermal activity (EDA). The mean amplitude of evoked EDA decreased from 2159+/-700 to 1413+/-515 microV following penile prosthesis surgery (P=0.017). The decrease in the amplitude of penile-evoked EDA following penile prosthesis implantation suggests the contribution of cavernous smooth muscle activity to the sudomotor responses prior to operation. Although corpus cavernosum sympathetic activity contributes to the penile skin recordings, these recordings are mostly the result of penile skin sudomotor sympathetic activity. Therefore, surface potentials recorded from penile skin should not be used for the evaluation of autonomic innervation of corpus cavernosum.     

携带ROCK2基因siRNA有效片段的慢病毒载体的筛选

目的:筛选能够显著抑制自发性高血压大鼠(SHR)阴茎海绵体平滑肌细胞内ROCK2基因表达的携带ROCK2基因siRNA的慢病毒载体。方法:设计并合成4个靶向ROCK2的siRNA片段,构建并包装成慢病毒载体。随机将5只SHR阴茎海绵体平滑肌细胞分为6组,每组每个样本3×104个细胞,每组5个样本,分别为:A组(未转染对照组)、B组(携带慢病毒转染组)、C~F组(分别携带靶向ROCK2基因siRNA 1~4号靶点的慢病毒转染组),以感染复(MOI)=80转染SHR阴茎海绵体平滑肌细胞,转染后48 h荧光显微镜下观察细胞GFP表达情况,并用RT-PCR检测各组被转染细胞ROCK2 mRNA的表达。结果:荧光显微镜下观察各组细胞转染效率均50%。与A组相比,B组ROCK2 mRNA的表达无明显改变(P﹥0.05);C、D、F组SHR阴茎海绵体平滑肌细胞ROCK2基因mRNA的表达较A组极显著下降(P0.01),抑制效率分别达到(43.91±8.19)%、(47.15±6.64)%、(25.7±6.03)%;E组SHR阴茎海绵体平滑肌细胞ROCK2基因mRNA的表达较A组显著下降(P0.05),抑制效率为(16.81±5.94)%。结论:本研究构建的4种携带ROCK2基因的siRNA慢病毒载体均能够显著抑制SHR阴茎海绵体平滑肌细胞内ROCK2基因的表达,其中有1种慢病毒载体抑制作用最强。     

Possible role of human growth hormone in penile erection

PURPOSE: Treatment with recombinant human growth hormone in adult patients with growth hormone deficiency increases nitric oxide and cyclic guanosine monophosphate (cGMP). We examined the functional in vitro effects of recombinant human growth hormone on tissue tension and cyclic nucleotide levels of human corpus cavernosum and detected changes in growth hormone in the cavernous and peripheral blood during different phases of penile erection. MATERIALS AND METHODS: Relaxant responses of human corpus cavernosum were investigated using the organ bath technique. Tissue levels of cGMP were determined by a specific radioimmunoassay after dose dependent exposition of isolated human corpus cavernosum strips to recombinant human growth hormone. In 35 healthy potent volunteers blood samples were obtained simultaneously from the corpus cavernosum and cubital vein during different functional conditions of the penis, including flaccidity, tumescence, rigidity and detumescence. Penile erection was induced by audiovisual and tactile stimulation. Serum growth hormone was determined by an immunoradiometric assay. RESULTS: Recombinant human growth hormone elicited dose dependent relaxation of human corpus cavernosum strips in vitro. The relaxing potency of recombinant human growth hormone was paralleled by its ability to elevate intracellular levels of cGMP. In vivo the peripheral growth hormone serum profile of the respective penile conditions did not significantly differ from those of cavernous serum. The main increase in growth hormone to greater than 90% was determined during developing penile tumescence, followed by a transient decrease afterward. CONCLUSIONS: These results suggest that penile erection may probably be induced by growth hormone through its cGMP stimulating activity on human corpus cavernosum smooth muscle.