Concentration of elastic system fibers in the corpus cavernosum, corpus spongiosum, and tunica albuginea in the rabbit penis

The corpus cavernosum (CC) extracellular matrix is essential for normal penile erection and is implicated in erectile dysfunction. Although investigations of these issues have used the rabbit CC, organization of its components is not well known to date. We characterized and quantified the volumetric density (Vv) of the elastic system fibers in the corpus spongiosum (CS), CC and tunica albuginea (TA) of the rabbit penis. Adult New Zealand rabbits (n = 10) were used. The penile mid-shaft fragments were fixed with 4% phosphate-buffered formalin solution and/or Bouin's liquid for 24-48 h, and processed using standard histological techniques. The sections were stained with Weigert's Fucsin-Resorcin with previous oxidation. The elastic system fibers Vv (%) was determined in 25 random fields of each fragment, using the M-42 test grid. The histochemical methods detected elastic system fibers in CS, CC and TA of all animals. The Vv of elastic fibers average was 25.03+/-2.0% for CC, 32.23+/-1.41% for CS and 22.38+/-3.61% for TA. Results for CC and CS were not significantly different. The great amount of elastic fibers distribution beneath the endothelium suggest that these fibers may have an important role in the erection process in rabbits. The present data should therefore provide important information for devising experiments and interpreting results when using the rabbit penis as a model for penile dysfunctions, especially when making comparisons with humans.     

阴茎海绵体平滑肌细胞培养进展

阐述阴茎海绵体平滑肌细胞（CCSM）培养对目前研究勃起功能障碍的重要性。比较CCSM与其他平滑肌细胞培养的差异,同时比较两种不同的CCSM培养方法结果的异同,为研究勃起功能障碍培养CCSM方法的选择提供参考。     

SD大鼠阴茎平滑肌细胞的获取和体外培养

目的 研究SD大鼠阴茎平滑肌细胞体外培养方法和生物学特性.方法 采用贴壁组织块培养法和酶消化培养法,对SD大鼠阴茎海绵体平滑肌细胞进行活细胞观察.结果 (1)SD大鼠阴茎海绵体平滑肌细胞为梭形,呈长轴平行排列,具有明显的方向性;体外贴壁快,生长迅速,体外培养的阴茎海绵体平滑肌细胞在合适的传代条件和比例下能够生存并保持其稳定的生物学特性.(2)酶消化培养法获得细胞的纯度高,接种后细胞生长快.贴壁组织块培养法操作简单易于掌握.结论 贴壁组织块培养法和酶消化培养法均可以获得平滑肌细胞,方法均方便可行.我们可根据实验的需要选择不同的方法.     

兔阴茎海绵体平滑肌细胞的快速分离及鉴定

目的探讨兔阴茎海绵体平滑肌细胞快速分离方法,为应用膜片钳技术研究阴茎勃起机制提供实验材料.方法采用木瓜蛋白酶和胶原酶两步酶解消化法,快速分离出新西兰大白兔阴茎海绵体平滑肌细胞并应用免疫组化鉴定.结果分离的细胞成活率较高,贴壁呈长梭形,胞膜光滑完整,胞浆均匀,可用于膜片钳记录.免疫组化鉴定为兔阴茎海绵体平滑肌细胞.结论酶解消化快速分离兔阴茎海綿体平滑肌细胞为全细胞膜片钳技术研究阴茎勃起功能障碍的电生理机制提供了较好的实验材料.     

Effects of sense tankyrase transfection on smooth muscle cells of corpus cavernosum in rat

This study was conducted to construct the eukaryotic expression vectors for sense tankyrase and to investigate the effects of tankyrase transfection on smooth muscle cells of corpus cavernosum in the rat. After the eukaryotic expression vectors for sense tankyrase were constructed and identified smooth muscle cells of rat corpus cavernosum were transfected with the recombinant plasmids of sense tankyrase (pcDNA-TNKS). Levels of DNA and RNA were then evaluated. Measurement of telomerase activity was conducted by TRAP-ELISA assay, the length of telomere by Southern blot and the growth curve by MTT assay. We have found that the eukaryotic expression vectors for sense tankyrase were constructed and the recombinant plasmids of sense tankyrase were transfected into smooth muscle cells of rat corpus cavernosum successfully; no significant differences in telomerase activity were observed between TNKS-transfected cells (SMC-TANKS), zero-load- transfected cells (SMC-Zeo), and non-transfected cells (SMC) (P > 0.05). The length of telomere in SMC-TANKS was longer than that in SMC-Zeo or SMC (P < 0.01), and the OD value of TNKS-transfected cell was significantly higher than that of the non-transfected cells (P < 0.01). These results suggested that the eukaryotic expression vectors for sense tankyrase were constructed successfully, which provides the basis for gene therapy. Transfection of recombinant plasmids of sense tankyrase helps change the telomerase length of smooth muscle cells of corpus cavernosum and extend the cell life span.     

人阴茎海绵体平滑肌细胞的原代培养及鉴定

目的　为研究人阴茎勃起功能提供方便的实验材料。　方法　取新鲜尸体阴茎海绵体平滑肌细胞 ,用含 2 0 %人AB型血清的DMEM液作体外原代培养 ,并以免疫组化法鉴定。　结果　经 10～ 14d培养后 ,6个培养瓶内均铺满梭状细胞 ,免疫组化法鉴定确认为人阴茎海绵体平滑肌细胞。　结论　人阴茎海绵体平滑肌细胞可在体外条件下生长传代 ,并可能成系 ,为阴茎勃起功能的研究提供方便的材料     

腺病毒介导的激活大鼠阴茎海绵体平滑肌细胞中NO／cGMP通路的实验研究

目的：探讨靶向大鼠iNOS基因的shRNA重组腺病毒载体对大鼠阴茎海绵体平滑肌细胞iN0s基因的激活作用,为阴茎勃起功能障碍（ED）的基因治疗提供实验依据。方法：将前期构建的重组腺病毒AdS—iN—OSrshRNA-EGFP（AdU6／shiNOS）和对照病毒AdU6／shControl,分别转染大鼠阴茎海绵体平滑肌细胞,分别在不同病毒MOI（25,50,75）值下72小时后采样检测。采用realtimeRT-PCR半定量检测AdU6／shiNOS对细胞iNOS基因mRNA表达影响;Western—blot法检测海绵体平滑肌细胞iNOS蛋白表达变化。然后培养基中加L—Arg（10mmol／L）,用酶联免疫法检测病毒转染72小时后海绵体平滑肌细胞内cGMP的浓度变化,记录AdU6／shiNOS对平滑肌细胞内cGMP的影响。结果：AdU6／shiNOS转染大鼠阴茎海绵体平滑肌细胞72小时后,和空白对照组、阴性对照组相比iN0s基因在mRNA和蛋白表达水平均显著上调（P〈O．05）,呈剂量依赖性,MOI一75时RNAa效果最好。而且转染72小时后,实验组原代平滑肌细胞内cGMP水平显著高于对照组及空白组（Pd0．05）。结论：利用腺病毒介导的RNAa技术,提高海绵体平滑肌细胞iN0s基因表达获得成功,可以增加阴茎海绵体平滑肌细胞cGMP水平,激活了NO／cGMP通路,这为勃起功能障碍的基因治疗研究开辟了新的方向。     

人阴茎海绵体平滑肌细胞三维培养模型的建立

目的 观察人阴茎海绵体平滑肌细胞(hCCMCs)在三维培养系统中的生物学特性.方法 体外分离hCCMCs,并用差速贴壁法进行纯化.免疫组化的方法检测α-平滑肌肌动蛋白(α-SMA)的表达,进行hCCMCs的鉴定.在单层贴壁培养的基础上,将hCCMCs在胶原凝胶中培养,形成三维培养系统,并对hCCMCs的形态结构、增殖情况进行研究.结果 原代培养的hCCMCs细胞形态不均一,经过差速贴壁法纯化后,细胞形态均一,免疫组化显示大多数细胞α-SMA阳性表达.三维培养系统中,hCCMCs在不同层面上呈三维生长.hCCMCs在三维培养系统中培养10d后,细胞数量无明显增殖(P>O.05),明显低于贴壁培养条件下细胞增殖速率(P<0.01).结论 hCCMCs三维培养系统可以观察hCCMCs在三维条件下的生长、增殖状况,研究hCCMCs与微环境的相互作用关系,有望为相关研究找到一种更为简单、可控性更强的体内实验替代方法.     

短发夹RNA对大鼠阴茎海绵体平滑肌细胞磷酸二酯酶5型基因表达的抑制作用

目的 观察短发夹RNA(shRNA)对大鼠阴茎海绵体平滑肌细胞磷酸二酯酶5型(PDE5)基因表达的抑制作用,探讨运用RNA干扰(RNAi)技术治疗勃起功能障碍(ED)的可行性.方法 构建靶向大鼠PDE5基因的shRNA重组腺病毒rAd-rPDE5-shRNA,将其转染大鼠阴茎海绵体平滑肌细胞48 h后,通过荧光标签进行显微计数确定转染效率,并以逆转录-聚合酶链反应(RT-PCR)及Western blot检测PDE5基因的表达水平.结果 rAd-rPDE5-shRNA构建成功,转染大鼠阴茎海绵体平滑肌细胞效率达95%以上,并使PDE5基因表达在mRNA水平抑制(80.78±2.30)%,在蛋白水平抑制(67.39±3.33)%.结论 以腺病毒为载体表达的shRNA能稳定、有效地抑制大鼠阴茎海绵体平滑肌细胞PDE5基因的表达.     

红景天苷对低氧环境下大鼠阴茎海绵体平滑肌细胞α-肌动蛋白和骨桥蛋白表达的影响

目的:探讨红景天苷对低氧SD大鼠阴茎海绵体平滑肌细胞(CCSMC)α-肌动蛋白、骨桥蛋白(OPN)表达的影响。方法:体外培养SD大鼠CCSMC,免疫组化法鉴定细胞;设正常对照组(21%O2浓度)、低氧组(1%O2浓度)、低氧+红景天苷1 mg/L组、低氧+红景天苷3 mg/L组、低氧+红景天苷5 mg/L组、低氧+前列腺素E1(PGE1)0.4μg/L组,分别培养48 h;RT-PCR法分别测定各组α-肌动蛋白、OPN的相对表达量。结果:体外培养的CCSMC生长良好,抗平滑肌α-肌动蛋白单克隆抗体免疫组化染色阳性;与正常对照组比较,低氧组细胞的α-肌动蛋白表达量下降、OPN表达量升高(P<0.01);与低氧组比较,红景天苷5 mg/L组α-肌动蛋白表达量升高、OPN表达量降低(P<0.01),与PGE1作用相当(P﹥0.05)。结论:低氧可引起SD大鼠CCSMC收缩型标志物α-肌动蛋白表达降低,合成型标志物OPN表达升高,推测低氧可能引起CCSMC由收缩型向合成型转化。红景天苷能抑制低氧引起的CCSMCα-肌动蛋白表达降低、OPN表达升高,浓度为5 mg/L时与PGE1作用几乎相当。     

Effects of castration on nitric oxide-mediated relaxations in male rat corpus cavernosum smooth muscle

BACKGROUND: The effects of castration on nitric oxide- mediated relaxations and nitric oxide synthase activity in male rat corpus cavernosum smooth muscles. METHODS: Eight-week-old male rats were assigned to two groups: control (sham operated) and castrated animals. After 8 weeks, corpus cavernosum smooth muscle strips were mounted in an organ bath for isometric tension recordings. Electrical field stimulation (EFS) was applied to the strips precontracted with 30 microM phenylephrine. The microdialysis probe was inserted into the strip, and Krebs-Henseleit solution was perfused into the probe. The dialysate during EFS and cholinergic stimulation was collected, and the amount of NO(-)(2)/NO(-)(3) (NOx) released in the dialysate was measured by the Greiss method. Sodium nitroprusside and carbachol were cumulatively added to the strips precontracted with 30 microM phenylephrine. RESULTS: EFS caused frequency-dependent relaxations and NOx releases in the strips. Pretreatment with N(omega)-nitro-L-arginine (100 microM) and tetrodotoxin (1 microM) completely inhibited relaxations and NOx releases. The maximum relaxation in the castration group was significantly greater than that in the control group. The release of NOx was significantly greater in the castration group than in the control group. Sodium nitroprusside relaxed the tissues in both groups similarly. Carbachol failed either to relax the tissue or to increase the amount of NOx production in the tissue. CONCLUSION: The present data suggest that castration enhances nitric oxide synthase activity and nitric oxide-mediated relaxations in the male rat corpus cavernosum.     

Effects of diabetes on nitric oxide-mediated relaxations in male rat corpus cavernosum smooth muscle

INTRODUCTION: We evaluated the effects of diabetes on nitric oxide-mediated relaxations and nitric oxide synthase activity in male rat corpus cavernosum smooth muscles. METHODS: Eight-week-old male rats were assigned to three groups: control (injected with the vehicle), DM (diabetes mellitus, induced by injection with 65 mg/kg streptozotocin), and TES (testosterone, testosterone supplemented after induction of diabetes). After 8 weeks, corpus cavernosum smooth muscle strips were mounted in an organ bath for isometric tension recordings. Electrical field stimulation (EFS, 2-ms pulse duration, 0.3-20 Hz and 3 s train) was applied to the strips precontracted with 30 microM phenylephrine. The microdialysis probe was inserted into the strip, and Krebs-Henseleit solution was perfused into the probe. The dialysate during EFS was collected, and the amount of NO(-)(2)/NO(-)(3) (NOx) released in the dialysate was measured by the Greiss method. Sodium nitroprusside (0.1 nM to 10 mM) and carbachol (1 nM to 10 mM) were cumulatively added to the strips precontracted with 30 microM phenylephrine. RESULTS: EFS caused frequency-dependent relaxations and NOx releases of the strips. Pretreatment with N(omega)-nitro-L-arginine (100 microM) and tetrodotoxin (1 microM) completely inhibited the relaxations and NOx releases. The maximum relaxation was significantly greater in the DM group than in the control or TES group. The release of NOx was significantly greater in the DM group than in the control or TES group. Sodium nitroprusside, the endothelium-independent vasodilator, relaxed the tissues in all three groups. There were no significant differences among control, DM and TES groups in the maximum relaxation to sodium nitroprusside. CONCLUSION: The present data suggest that diabetes enhances nitric oxide synthase activity and nitric oxide-mediated relaxations in the male rat corpus cavernosum by the reduced testosterone level in the diabetic animals.     

Leiomyoma of the corpus cavernosum of the penis

While soft tissue tumors can occur in the penis, corpus cavernous tumors are rare. Reported cases of corpus cavernous tumors are from metastases of advanced malignancy, such as cancers of the bladder, prostate, rectosigmoid colon, kidney, pancreas, liver, testis and nasopharynx. Primary corpus cavernous tumors are extremely rare and have possibly never been reported before. Herein, we report a case of leiomyoma of the corpus cavernosum. After the diagnosis of leiomyoma was established, total excision of the tumor was not attempted and the tumor remained unchanged in size and shape over a follow-up period of 15 months.     

Potential differentiation of human mesenchymal stem cell transplanted in rat corpus cavernosum toward endothelial or smooth muscle cells

One of the causes of erectile dysfunction (ED) is the damaged penile cavernous smooth muscle cells (SMCs) and sinus endothelial cells (ECs). To investigate the feasibility of applying immortalized human mesenchymal stem cells (MSCs) to penile cavernous ECs or SMCs repair in the treatment of ED, the in vivo potential differentiation of the immortalized human MSCs toward penile cavernous endothelial or smooth muscle was investigated. One clone of immortalized human bone marrow mesenchymal stem cell line B10 cells via retroviral vector encoding v-myc were transplanted into the cavernosum of the Sprague-Dawley rats and harvested 2 weeks later. The expression of CD31, von Willebrand factor (vWF), smooth muscle cell actin (SMA), calponin and desmin was determined immunohistochemically in rat penile cavernosum. Multipotency of B10 to adipogenic, osteogenic or chondrogenic differentiation was found. Expression of EC specific markers (CD31 or vWF protein) and expression of SMC specific markers (calponin, SMA or desmin protein) were demonstrated in grafted B10 cells. When human MSCs were transplanted into the penile cavernosum, they have the potential to differentiate toward ECs or SMCs. Human MSCs may be a good candidate in the treatment of penile cavernosum injury.     

Microsurgical revascularization of the canine corpus cavernosum penis

The concept of vasculogenic impotence in humans is addressed by attempting to surgically create and correct vasculogenic impotence in healthy dogs. Initial experience reveals that only by extensive pelvic vascular interruption can such impotence be created and that, at best, it is only temporary because of collateralization. Microsurgical revascularization using both direct femoral artery implantation and femoral vein interposition into the corpus cavernosum penis yielded low patency rates. Further studies on this technique are needed before it can be recommended for widespread use in human beings.     

Construction of cavernosum smooth muscle using umbilical artery smooth muscle cells seeded on acellular corporal collagen matrices

The objective of this study was to investigate the feasibility of tissue engineering of corpus cavernosal smooth muscle. Acellular corporal collagen matrices (ACCMs) were obtained from the penis of adult rabbits by a cell removal procedure. ACCMs were implanted into the back muscles of allogenic rabbits to investigate the resulting immunological reaction. Human umbilical artery smooth muscle cells (HUASMCs) were isolated from human umbilical arteries through explant techniques and expanded in vitro . Subsequently, third and fifth passage HUASMCs were seeded to ACCMs at a concentration of 30 × 10⁶ cells/mL. Then, seeded ACCMs were implanted subcutaneously in athymic mice. The implants were retrieved at 10, 20 and 40 days after implantation. Histochemistry, immunohistochemistry and scanning electron microscopy were performed to analyse the morphological characteristics of the engineered tissues. Additionally, organ bath studies were performed to address the contractility of the engineered tissues. The decellularization process successfully extracted all cellular components while preserving the original collagen fibers. The immunological reaction to ACCMs consisted of only a transient nonspecific inflammatory response. Light and scanning electron microscopy demonstrated that HUASMCs extended onto the three-dimensional ACCMs scaffolds in vitro . Histological analyses of the explants from all time points demonstrated a progressive regeneration of smooth muscle, with structures very similar to native corpus cavernosum smooth muscle. The maximum contraction force induced by phenylephrine and electrical stimulation were 3.64 ± 0.18 g/100 mg and 2.50 ± 0.21 g/100 mg, respectively. Our study demonstrates that HUASMCs can be seeded on three-dimensional ACCM scaffolds and will develop tissues similar to that of the native corpus cavernosum smooth muscle.     

Expression of myosin isoforms in the smooth muscle of human corpus cavernosum

The molecular interaction between smooth muscle (SM) myosin and actin in the corpus cavernosum (CC) determines the erectile state of the penis. A key mechanism regulating this interaction and subsequent development and maintenance of force is alternative splicing of SM myosin heavy chain (MHC) and 17 kDa essential SM myosin light chain (MLC) pre-mRNAs. Our aim was to examine the relative SM myosin isoform composition in human CC. Tissue samples were obtained from 18 patients with erectile dysfunction (ED), Peyronie's disease, or both. One specimen was obtained during a transgender operation. Patients then were stratified according to presence of diabetes mellitus, hypertension, ED, or Peyronie's disease, as well as failure of phosphodiesterase-5 (PDE5) inhibitors and history of previous pelvic or penile surgeries, radiation, or both. Our results revealed that all human CC samples expressed only the SM-A isoform. There was a predominance of SM2 isoform mRNA relative to SM1 across all samples, with a mean of 63.8%, which correlated with protein analysis by gel electrophoresis. A statistically significant difference was found between patients who had undergone previous pelvic surgery, radiation, or both and those who did not. The ratio of LC(17b) to LC(17a) was approximately 1:1 for all patients, with a mean of 48.9% LC(17b). Statistical difference was seen in the relative ratio of LC(17b) to LC(17a) among the group who failed conservative therapy with PDE5 inhibitors compared with all others. In conclusion, we determined the SM myosin isoform composition of human CC and present for the first time differences in relative myosin isoform expression among patients with several risk factors contributing to their cause of ED. Our data reflect the fact that alternative splicing events in the MHC and 17 kDa MLC pre-mRNA may be a possible molecular mechanism involved in the altered contractility of the CCSM in patients with ED.     

Effect of TCDD on corpus cavernosum histology and smooth muscle physiology

A polyhalogenated aromatic hydrocarbon, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is one of the most potent toxic environmental pollutants. Decreases in spermatogenesis and the ability to conceive and carry a pregnancy to term are the most sensitive signs of reproductive toxicity by TCDD in the mammal, but no report of its effect on the erectile function exists. We performed this study to investigate the effect of TCDD on the erectile function. New Zealand white rabbits were treated intraperitoneally with 1 microg/kg of TCDD. At 4 (Gr I) and 8 (Gr II) weeks after the administration of TCDD, cavernosal tissues were harvested for strip study in the organ bath and testes were prepared for histologic examination. Compared to the maximal amplitude of 17.1+/-4.12 mN in normal control (Gr III), the contractions to cumulative concentrations of NE (10(-8)-10(-4) M) were significantly decreased to 6.57+/-1.34 and 5.45+/-1.01 mN in Groups I and II, respectively. Compared to 51.12+/-7.38% in Gr III, relaxation to cumulative concentration (10(-8)-10(-4) M) of acetylcholine was significantly decreased to 17.25+/-2.17% (Gr I) and 9.73+/-2.17% (Gr II) at a concentration of 10(-4) M, respectively. Compared to 75.12+/-13.18% in Gr III, relaxation to cumulative concentration (10(-8)-10(-4) M) of SNP was significantly decreased to 31.49+/-7.89% (Gr I) and 18.54+/-6.12% (Gr II) at a concentration of 10(-4) M, respectively. Histologically, intracavernosal fibrosis, abnormal subtunical deposition of fat and decreased sinusoidal space with consequent increase of trabecular smooth muscle contents were identified in TCDD-treated groups. In TCDD-treated animals, seminiferous tubules showed a decrease of germ cells with vacuolar degeneration and apoptotic cells. Spermatids were hardly seen. These results suggest that TCDD inhibits spermatogenesis and has a potential harmful effect on erectile function via changes of corpus cavernosum histology and smooth muscle physiology.