Fibrosis of corpus cavernosum in animals following cavernous nerve ablation

Aim: To investigate alterations of smooth muscle celis and collagen fibers in corpus cavernosum following cavernous neurectomy and its relation to the expression of transforming growth factor-β1 (TGF-β1). Methods: Ten adult male SD rats (neurectomy group) were subject to a bilateral cavernous nerve (CN) resection aseptically under an operating microscope, with 6 sham-operated rats as the control. Fifteen weeks after the operation, the penile speci mens were collected and prepared for quantitative-analyzing of ratio of smooth muscle to collagen fibers in corpus cavernosum with confocal microscopy, and for detecting the expression of TGF-β1 by RT-PCR and western-blot. Resulte: Smooth muscle celis that show red color after fluorescent-labeling with tetramethylrhodamine isothiocyanate phalloidin and collagen fibers that produce green autofluorescence after paraformaldehyde fixation were clearly iden tified     

复合雪旺细胞的猪小肠粘膜下层对大鼠海绵体神经损伤勃起功能恢复的实验研究

目的:研究雪旺细胞(SCs)与猪小肠粘膜下层(SIS)构成的人工神经移植替代损伤的大鼠双侧海绵体神经(CN)恢复大鼠的勃起功能。方法:体外培养SCs并复合于SIS,成功制备成SIS与SCs的复合体,选取健康SD大鼠33只,随机分为3组:假手术组(n=11)、CN切断组(n=11)、SCs+SIS组(n=11)。各组大鼠于术后3个月进行阿朴吗啡试验;然后取阴茎海绵体中后部海绵体组织,进行nNOS免疫组织化学染色。结果:①联合使用机械剥离、混合酶消化、差速贴壁法、Ara-C短期使用等方法可获得较高纯度的SCs,满足神经组织工程的需要;②经过机械刮除、化学方法消毒制备的SIS作为一种天然支架材料,与SCs具有良好的细胞生物相容性,适合SCs在其表面粘附、生长、增殖和分化;③术后3个月行阿朴吗啡试验:无论是勃起率还是勃起次数,SCs+SIS组均显著高于CN切断组(P<0.01),但低于假手术组(P<0.01);④nNOS神经纤维数目:SCs+SIS组与CN切断组差异有统计学意义(P<0.01),但二者均低于假手术组(P<0.01)。结论:SCs+SIS作为神经移植物可以修复缺损的CN,并在一定程度上恢复阴茎勃起功能。     

Pentoxifylline promotes recovery of erectile function in a rat model of postprostatectomy erectile dysfunction

Background

Cavernous nerve (CN) injury during radical prostatectomy (RP) causes CN degeneration and secondary penile fibrosis and smooth muscle cell (SMC) apoptosis. Pentoxifylline (PTX) is a phosphodiesterase inhibitor that further inhibits multiple cytokine pathways involved in nerve degeneration, apoptosis, and fibrosis.

Objectives

To evaluate whether PTX enhances erectile function in a rat model of CN injury.

Design, Setting and Interventions

Forty male Sprague-Dawley rats underwent CN crush injury and were randomized to oral gavage feeding of phosphate-buffered saline (vehicle) or PTX 25, PTX 50, or PTX 100 mg/kg per day. Ten animals underwent sham surgery and received vehicle treatment. Treatment continued for 28 d, followed by a wash-out period of 72 h. An additional eight rats underwent resection of the major pelvic ganglion (MPG) for tissue culture and examination of direct effects of PTX on neurite sprouting.

Measurements

Intracavernous pressure recording on CN electrostimulation, immunohistologic examination of the penis and the CN distal to the injury site, and length of neurite sprouts in MPG culture.

Results

Daily oral gavage feeding of PTX resulted in significant improvement of erectile function compared to vehicle treatment in all treated groups. After treatment with PTX 50 and PTX 100 mg/kg per day, the expression of neuronal nitric oxide synthase in the dorsal penile nerve was significantly higher than in vehicle-treated rats. Furthermore, PTX treatment prevented collagen deposition and SMC loss in the corpus cavernosum. In the CN, signs of Wallerian degeneration were ameliorated by PTX treatment. MPG culture in medium containing PTX resulted in a significant increase of neurite length.

Conclusions

PTX treatment following CN injury in rats improved erectile recovery, enhanced nerve regeneration, and preserved the corpus cavernosum microarchitecture. The clinical availability of this compound merits application in penile rehabilitation studies following RP in the near future.     

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.     

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.     

Effects of chronic renal failure on the expression of connexin 43 in the rat＇s corpus cavernosum

Aim： To explore the mechanism of chronic renal failure （CRF）-related erectile dysfunction （ED）. Methods： CRF experimental models were established by 5/6 nephrectomy from male Sprague-Dawley rats. Both the rats from the control group （NCRF group, n = 6） and the experimental group （CRF group, n = 30） were injected with a low dose （80 μg/g） of apomorphine in the 12th week after resection surgery to measure corresponding penile erections. Western blot method was thereafter conducted to measure the expression of connexin 43 （CX43） in the rat corpus cavernosum in the 12th week after the resection surgery. Results： There was one death in the NCRF group and five in the CRF group. The penile erection ratio of the CRF group was 28% （7/25）, whereas that of the NCRF group was 100% （5/5）, which presents a significant difference between the two groups （P 〈 0.05）. In terms of penile erection frequency, the average of the CRF group was 1.0 ± 0.0, which was significantly different from that of the NCRF group （2.2 ± 0.8） （P 〈 0.05）. As for the expression of CX43 in the rat corpus cavernosum, a notable difference existed between the CRF group （0.21 ± 0.07） and the NCRF group （0.53 ± 0.27） （P 〈 0.01）. Conclusion： CRF significantly reduces the erectile function of rats. A close correlation exists between the expression of CX43 in rats＇ corpus cavernosum and CRF-related ED. （Asian J Andro12008 Mar; 10： 286-289）     

Proteomic analysis of rat penile tissue in a model of erectile dysfunction after radical prostatectomy

OBJECTIVE: To identify differential protein expression in penile tissue in a rat model of erectile dysfunction (ED) at an early stage after bilateral cavernosal nerve (CN) neurectomy, using proteomic techniques. MATERIALS AND METHODS: Twelve male adult Sprague-Dawley rats were randomly divided into two equal groups, one having bilateral CN resection and one a control group. The penises were harvested 7 days after CN resection. Total protein was separated into >1250 protein spots by two-dimensional electrophoresis using pH 3-10 nonlinear immobilized pH gradient strips. Differential expression of proteins was analysed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and database searching. RESULTS: Thirty-two proteins were significantly changed in the denervated penis, of which 25 (including nine up-regulated and 16 down-regulated) with cytoskeletal functions, and pathophysiological functions related to energy metabolism and oxidative stress, were identified. Examples include transgelin, creatine kinase B, annexin-1 and galactin-7. CONCLUSIONS: The expression of several important proteins participating in pathophysiological processes of penile tissue are changed early after bilateral CN neurectomy. These changes might give new insights into the cellular and molecular mechanisms involved in neurogenic ED development, and indicate potential therapeutic targets.     

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     

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.     

2型糖尿病大鼠血浆同型半胱氨酸与阴茎海绵体内NOS和内源性CO的相关性研究

目的:研究2型糖尿病性大鼠血浆同型半胱氨酸(Hcy)与阴茎海绵体内NOS和内源性CO的相关性。方法:选取3月龄雄性Wistar大鼠50只,随机选取10只为对照组(A组);高糖高脂饲料饲养4周后从其他40只大鼠中筛选出30只构建成功的糖尿病(DM)大鼠模型,随机分成3组:DM大鼠组(B组);胰岛素治疗组(C组)和叶酸+维生素B12治疗组(D组)。8周及12周后注射阿朴吗啡观察各组大鼠阴茎勃起情况。12周后测各组大鼠血浆总Hcy含量及阴茎海绵体内NOS活性和CO含量。结果:与A组比较,B组大鼠血浆Hcy浓度明显升高,阴茎勃起功能明显降低,阴茎海绵体NOS活性和CO含量均下降,差异有显著性(P<0.01)。2型DM大鼠中高Hcy血症发生率为55%。与B组比较,C组和D组中大鼠血浆Hcy浓度显著下降,阴茎勃起功能、阴茎海绵体NOS活性均升高(P<0.01),Hcy与NOS(rA=-0.89,rB=-0.76,rC=-0.91,rD=-0.91)及CO含量(rA=-0.82,rB=-0.77,rC=-0.93,rD=-0.81)均呈负相关。结论:2型DM大鼠血浆中的高Hcy可能是引起阴茎海绵体NOS活性下降、CO含量下降,进而导致DM ED发病的分子机制之一。胰岛素、叶酸和维生素B12可以改善DM大鼠的勃起功能,提高阴茎海绵体NOS活性和CO含量。     

Characterization of corpus cavernosum smooth muscle cell phenotype in diabetic rats with erectile dysfunction

Phenotypic modulation from a contractile to a proliferative state within vascular smooth muscle cells has a critical role in the pathogenesis of a variety of cardiovascular diseases. To investigate the characterization of corpus cavernosum smooth muscle cell phenotype in diabetic rats with erectile dysfunction, a group of Sprague-Dawley rats (n=30) were induced by intraperitoneal injection of streptozotocin (60?mg?kg(-1)) and screened by subcutaneous injection of apomorphine (100?μg?kg(-1)) for the measurement and comparison of the penile erections, and then three different groups were defined. Primary corpus cavernosum smooth muscle cells were cultured and passaged. The cavernous tissue segments were subjected to quantitative real-time polymerase chain reaction to determine the expressions of smooth muscle α-actin (SMA), SM myosin heavy chain (SMMHC), smoothelin, calponin and myocardin. Cell contractility in vitro and western blot analysis of SMA and SMMHC in the cavernous tissues and cells were determined. Compared with the control group (n=8) and the diabetes mellitus group (n=5), the expressions of SMA, calponin, SMMHC, smoothelin and myocardin mRNA were decreased in the cavernous tissues in rats of the diabetic erectile dysfunction group (n=15; P=0.001 and 0.02, P=0.014 and 0.012, both P<0.001, P=0.005 and <0.001, P=0.003 and 0.035, respectively). The levels of SMA and SMMHC proteins showed a significant decrease in cavernous tissues and cultured cells in rats of the diabetic erectile dysfunction group. Cells of the diabetic erectile dysfunction group exhibited significantly less contractility compared with those of other groups (P<0.001). Corpus cavernosum SM cell possesses the ability to modulate the phenotype under hyperglycemic conditions, which could have a key role in the pathogenesis of diabetic erectile dysfunction.     

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.     

前列腺癌根治术后勃起功能障碍动物模型的建立

目的 对大鼠海绵体神经(CN)进行解剖并对其造成钳夹损伤,以建立前列腺癌根治术(RP)后勃起功能障碍(ED)的动物模型.方法 将36只雄性SD大鼠随机分为模型组、假手术组、正常组,术后4周通过阿朴吗啡(APO)实验及海绵体内压(ICP)/平均动脉压(MAP)测定、阴茎神经型一氧化氮合酶(nNOS)免疫组织化学检测以评估建立模型的效果.结果 术后4周3组大鼠的阴茎勃起率和平均勃起次数分别为0%和0次、100%(11/11)和(2.24±0.86)次、100%(12/12)和(2.39±0.92)次.模型组大鼠无明显勃起反应,假手术组、正常组可见明确勃起反应.电刺激盆腔星状神经节(MPG)前3组大鼠ICP/MAP分别为(0.13±0.04)、(0.10±0.03)、(0.12±0.03),电刺激MPG后分别为(0.12±0.05)、(0.57±0.08)、(0.58±0.06),电刺激MPG前3组ICP/MAP差异均无统计学意义(P＞0.05),模型组与假手术组、正常组的电刺激MPG后ICP/MAP差异有统计学意义(P＜0.05).3组大鼠阴茎nNOS阳性神经纤维数分别为(23.04±2.59)、(73.94±7.51)、(80.26±6.95),模型组大鼠阴茎nNOS阳性神经纤维数明显少于假手术组、正常组(P＜0.05).结论 大鼠CN结构与人类非常相似,CN钳夹损伤是建立RP术后ED模型的可靠的方法.

Abstract：

Objective To identify rat cavernous nerve (CN) and develop a rat model of erectile dysfunction (ED) after radical prostatectomy (RP) caused by injury of cavernous nerve.Methods Thirty-six male SD rats were were randomly divided into 3 groups:model group,sham-operation group and nomal group.Four weeks after surgery,rat models were evaluated by apomorphine test,ICP/MAP measurement and nNOS-positive nerve fibres in penis.Results The rats in model group had no erectilty in apomorphine test and electical stimulating MPG.The rats in sham-operation group and nomal group had erectility at the same conditions,accompanied with increased ICP/MAP in electrical stimulation ( P＜0.05 ).The nNOS-positive nerve fibres in model group ( 23.04 ± 2.59 ) were less than those in sham-operation group (73.94 ±7.51 ) and normal group (80.26 ±6.95 ).Conclusion Cavernous nerve in rats is highly similar to that in human beings.The rat model of erectile dysfunction after radical prostatectomy caused by cavernous nerve crush injury is reliable.     

Erektionsstatus nach nervenerhaltender radikaler Prostatektomie

The time lapse before recovery of erectile function after nerve-sparing radical prostatectomy is still under debate. Several pathophysiologies are postulated for postoperative erectile function rehabilitation. In prospective studies we measured nocturnal penile tumescence (NPTR) in the acute phase during the first night after catheter removal subsequent to nerve-sparing radical prostatectomy to assess the neuronal organic erectile integrity.Eighteen sexually active patients suffering from local prostate cancer underwent bilateral and unilateral nerve-sparing retropubic radical prostatectomy. All patients completed an IIEF-5 questionnaire concerning erectile function preoperatively. The transurethral catheter was removed 14 days after surgery, and nocturnal penile tumescence was measured with an erectometer (Rigi-Scan) in each patient during the following night. None of these patients received any comedication interacting with erectile function.The preoperative IIEF score was >18 in all patients. After catheter removal, 17 of 18 patients (95%) had nocturnal penile radial rigidity >70% that persisted for >10 min during one night. In a control of four patients without a nerve-sparing procedure, no nocturnal erections were recorded.The measurement of NPTR in the acute phase after nerve-sparing radical prostatectomy showed retained erectile function even during the "first" night after catheter removal. Our findings are important for an appropriate choice of pharmacotherapy for optimal recovery of erectile function. In cases of early penile erections, the cavernous nerve had been well preserved during surgery providing good neuronal integrity, and PDE-5 inhibitors can support organic rehabilitation of the corpus cavernosum. In the absence of early penile erections, the neuronal integrity of the cavernous nerve is presumed to be impaired. In this case, additional injection therapy should be chosen to support recovery of spontaneous erectile function.     

血红素氧合酶2在去势大鼠阴茎海绵体内的表达

目的:研究去势大鼠阴茎海绵体血红素氧合酶2(HO-2)和内皮型一氧化氮合酶(eNOS)的表达,探讨雄激素与HO-2、eNOS在ED中的作用及相关性。方法:10周龄雄性SD大鼠40只,分为4、8、12周组和正常对照组各10只,实验组采取手术切除双侧睾丸,对照组采取假手术。分别于术后4、8、12周测定大鼠血清睾酮(T)、阴茎海绵体内压(ICP)、平均颈动脉压(MAP),取阴茎标本,采用Western印迹分析阴茎海绵体HO-2含量,免疫组化分析HO-2和eNOS的表达。结果:去势各组血清T水平较正常对照组显著下降(P<0.05)。经3V、5V电压刺激后去势各组ICP/MAP值明显下降(P<0.05)。HO-2在正常和去势大鼠阴茎海绵体组织均有表达,去势4周组HO-2光密度分布曲线下面积(341.50±99.70)较正常组(876±443.36)和去势8周组(705.00±152.74)明显下降(P<0.05),去势8周与正常组之间无显著变化(P>0.05),去势12周没有检测到HO-2的表达。eNOS主要表达于阴茎海绵体血管内皮细胞,去势组eNOS(123.94±30.23)较正常组(421.21±125.12)差异有显著性(P<0.05)。T与eNOS和HO-2表达呈高度正相关(r=0.976、0.946,P均<0.05)。结论:雄激素可能通过影响大鼠阴茎海绵体HO-2、eNOS的表达参与阴茎勃起功能调控。     

可分泌表达人降钙素基因相关肽重组腺相关病毒对糖尿病大鼠阴茎勃起的作用

目的:探讨重组腺相关病毒(rAAV)介导人降钙素基因相关肽(hCGRP)基因转移在糖尿病大鼠阴茎海绵体平滑肌分泌表达及其对阴茎勃起的作用。方法:建立链佐脲菌素(STZ)糖尿病大鼠模型,随机分为3组,分别将VssHGCMV-hCGRP、VssCMV-GFP和rAAV空病毒液注射于阴茎海绵体。在注射后5 d,采用SMUP-PC型生物信号处理系统检测阴茎背神经电刺激诱发的阴茎勃起反应及海绵体内压(ICP)变化。切取海绵体组织,通过免疫组化技术和激光共聚焦显微镜分别检测hCGRP和绿色荧光蛋白(GFP)表达,以放射免疫法检测组织中环磷酸腺苷(cAMP)和环磷酸鸟苷(cGMP)变化。结果:在VssCMV-GFP转染后5 d,显示阴茎海绵体内几乎所有组织均有广泛的GFP表达,而rAAV空病毒转染的海绵体则无GFP表达。VssHGCMV-hCGRP转染STZ诱导的糖尿病大鼠后5d,电刺激阴茎背神经可诱发明显的阴茎勃起,监测ICP明显增高[(60.5±4.5)mm Hg,1 mm Hg=0.133kPa],而对rAAV空病毒转染的对照组STZ糖尿病大鼠以同样的参数电刺激阴茎背神经则无勃起反应,ICP无明显增加[(22.3±1.3)mm Hg],两组差异有显著性(P<0.01)。免疫组化观察显示在VssHGCMV-hCGRP转染的STZ糖尿病大鼠阴茎海绵体组织中hCGRP表达增强,同时当电刺激阴茎背神经诱发勃起反应时,海绵体内cAMP和cGMP水平均升高,分别为(48.4±6.5)nmol/L和(21.2±13.6)nmol/L,较rAAV空病毒组[(16.7±2.5)nmol/L和(0.42±0.12)nmol/L]明显增高(P<0.01)。结论:经阴茎海绵体内注射重组腺相关病毒VssHGCMV-hCGRP在糖尿病大鼠阴茎海绵体内获得了hCGRP转基因高效表达,其可增加阴茎背神经电刺激诱发的阴茎ICP和勃起反应。     

Neurotrophic effect of bone marrow mesenchymal stem cells for erectile dysfunction in diabetic rats

It has been demonstrated that intracavernous injection of bone marrow-derived mesenchymal stem cells (BM-MSCs) had beneficial effects on improving erectile function in type-1 diabetic rats. This study was designed to investigate the neurotrophic effect of BM-MSCs for type-1 diabetic rats. Streptozocin-induced type-1 diabetic rats were randomly divided into three groups: diabetic group, BM-MSCs-treated group and BM-MSCs-conditioned medium-treated group. At the 3d, 1 and 2w time points after BM-MSCs injection, three randomly selected rats in MSCs group were sacrificed and penile samples were harvested to detect BM-MSCs in penile tissue. Four weeks after intracavernous injection of BM-MSCs or BM-MSCs-conditioned medium, intracavernous pressure (ICP) was assessed to evaluate the erectile function. Immunohistochemistry was used to track labelled BM-MSCs in penile tissue and to detect neuronal nitric oxide synthase (nNOS) and neurofilament (NF) positive fibres in penile dorsal nerve. Enzyme lined immunosorbent assay (ELISA) was used to measure the concentrations of vascular endothelial growth factor (VEGF), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in BM-MSCs-conditioned medium. BM- MSCs secreted detectable levels of VEGF, BDNF and NGF. Intracavernous injection of BM-MSCs improved erectile function in diabetic rats. The functional improvement was accompanied by promoted nNOS and NF positive nerve fibres within penile dorsal nerve in type-1 diabetic rats. Histological data revealed a time-dependent decrease in the number of BM-MSCs in the corpus cavernosum following injection. Furthermore, the beneficial effect of BM-MSCs was partially repeated by BM-MSCs-conditioned medium. Intracavernous injection of BM-MSCs is effective in improving nerve regeneration in diabetic rats. Paracrine effects of BM-MSCs are probably involved in the improvement.     

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。