实验性高脂兔体外培养阴茎海绵体平滑肌细胞凋亡的研究

目的观察高脂诱导的血管性ED家兔阴茎海绵体平滑肌（Corpus Cavernosum Smooth Muscle，CCSM）细胞形态、结构变化及高脂对CCSM细胞凋亡、增殖的影响。方法雄性新西兰家兔10只，随机分为模型组和对照组，每组各5只。模型组为采用经高脂、高胆固醇饮食结合球囊导管扩张双侧髂内动脉建立、鉴定的血管性ED模型；对照组喂以正常饮食。8周后切取阴茎CCSM组织观察并做体外细胞培养。经处理后分别在光镜、透射电镜下观察，流式细胞仪检测细胞凋亡，MTT比色法观察细胞增殖活性，RT-PCR法研究Caspase-3 mRNA的表达情况，免疫荧光法检测Caspase-3活性。结果与对照组相比，模型组光镜和电镜下CCSM细胞内含较多脂质囊泡，线粒体、内质网等细胞器出现损伤，凋亡小体明显增多。流式细胞仪检测证实模型组CCSM细胞凋亡率较对照组明显增高（1．44±0．20％Vs5．96±1．60％，P〈0．01）。MTT比色法显示高脂对CCSM的增殖有明显抑制作用（模型组与对照组A值分别为：0．52±0．08Vs1．42±0．04，P〈0．01）。模型组的Caspase-3 mRNA表达和活性值均显著高于对照组（0．74±0．00Vs0．65±0．00，P〈0．01；活性值0．03±0．00Vs0．01±0．00，P〈0．05）。结论高脂、高胆固醇饮食可严重影响CCSM结构，促进CCSM细胞凋亡并抑制其增殖。CCSM细胞凋亡／增生的失衡可能是高脂导致ED发生发展的重要病理环节。     

海绵体平滑肌细胞的体外培养的研究

外生殖器(主要指雄性的阴茎,雌性的阴蒂)勃起是性生活的重要一环,海绵体平滑肌是勃起的重要因素,大多数的勃起功能障碍(erectile dysfunction,ED)与组织细胞的功能改变密切相关。目前的研究纷纷将海绵体平滑肌细胞(smooth muscle     

糖尿病大鼠阴茎海绵体细胞凋亡的实验研究

目的 观察糖尿病(DM)大鼠阴茎海绵体细胞凋亡,探讨DM性阴茎勃起功能障碍(ED)的发病机理。方法 本文通过四氧嘧啶(Alloxan,AXN)诱导的DM大鼠模型,饲养8周后处死动物并取阴茎海绵体。用琼脂糖凝胶电泳法进行DNA片段分析;用TUNEL法检测细胞凋亡。结果 DM组DNA片段分析出现凋亡特征性梯带。DM组较对照组勃起组织凋亡细胞数明显增多(P<0.01)。结论 DM大鼠阴茎海绵体凋亡细胞增加,细胞凋亡可能是DM性ED的发病机理。     

可卡因诱导大鼠阴茎细胞凋亡的研究

吸食可卡因所引起的各种医学和社会问题愈来愈引起人们的关注 ,我们通过动物实验探讨长期使用可卡因与阴茎细胞凋亡的关系。报告如下。材料与方法　雄性Ｗｉｓｔａｒ大鼠 6 0只 ,鼠龄 2 5ｄ。分实验组和对照组。从30ｄ开始 ,实验组每只大鼠每天皮下注射溶解于 0 .2ｍｌ蒸馏水的     

家兔阴茎海绵体平滑肌细胞体外培养方法对比研究

目的　探索更好、更方便的家兔阴茎海绵体平滑肌细胞体外培养方法。方法　分别采用组织块法、酶消化法、组织块 酶消化法对家兔阴茎海绵体平滑肌细胞进行培养 ,在倒置显微镜下对培养过程的细胞分别作了生长情况及形态学观察。用HE染色、MTT法分别描绘原代培养细胞的生长曲线、细胞分裂指数曲线 ;用倒置显微镜观察活细胞的贴壁过程 ,计数法测定细胞贴壁率。结果　组织块法及酶消化法培养的细胞其生长状况和形态学各有自己的特点。组织块法的细胞生长慢 ,培养时间相对较长 ,纯度相对较高 ,原代培养细胞生长速度快 ,但由于该法消化时间不易掌握 ,常影响其成功率 ,多数细胞呈梭形 ,细胞的密度高 ,原代培养时间为 7～ 10d。结论　每种培养方法都有各自的优缺点。我们可根据实验的需要而选择不同的方法或联合培养     

高脂对兔阴茎海绵体平滑肌细胞含量及结构的影响

目的 观察高脂诱导血管性ED家兔阴茎海绵体平滑肌细胞(smooth muscle cell, SMC)含量变化以及海绵体组织超微结构的改变情况.方法 8只性成熟雄性新西兰家兔随机分两组,每组各4只.对照组喂以正常饮食,模型组采用高脂、高胆固醇饮食结合球囊导管扩张双侧髂内动脉法建立血管性ED模型.第8周时切取阴茎海绵体组织,HE及特殊染色处理后在光镜、透射电镜下观察,计算机辅助彩色图像分析平滑肌及胶原构成百分比.RT-PCR法研究Caspase-3 mRNA的表达情况,免疫荧光法检测Caspase-3活性.结果 计算机分析表明模型组海绵体SMC含量较对照组明显减少(22.66±10.97)% vs (46.72±7.30)%,P<0.01.光镜和电镜下所见两组阴茎海绵体组织的超微结构呈显著差异.主要表现为模型组白膜结构改变,海绵体SMC明显减少,胶原成分增多.内皮细胞及SMC内存在脂质囊泡,线粒体、内质网等细胞器出现损伤,SMC凋亡小体明显增多.模型组的Caspase-3 mRNA.表达和活性值均显著高于对照组mRNA:(0.77+0.01) vs (0.68±0.01),P<0.01;活性值:(0.04±0.00) vs (0.02±0.00),P<0.05.结论 高脂、高胆固醇饮食可严重影响阴茎海绵体组织结构,主要表现为海绵体组织中SMC成分减少、胶原增加,使阴茎海绵体发生纤维化,这可能是高脂导致ED的重要原因之一.     

糖尿病大鼠阴茎组织细胞凋亡的研究

目的 研究阴茎海绵体组织细胞凋亡与糖尿病的关系。方法 腹腔内注射链脲佐菌素制备糖尿病大鼠模型，分别于第6、8周观察其阴茎勃起功能，并用TUNEL法检测细胞凋亡。结果 糖尿病组大鼠勃起功能明显下降，且细胞凋亡率高于对照组并随病程而增加。结论 糖尿病严重影响大鼠的勃起功能及增加海绵体组织细胞凋亡。细胞凋亡的增加可能是糖尿病性勃起功能障碍的发病机制之一。     

促进阴茎海绵体平滑肌松弛的植物性中药研究进展

勃起功能障碍 (ED)是中老年男性常见病及多发病。祖国医药治疗ED有着悠久的历史 ,但主要是临床经验研究 ,缺乏说服力。近年来 ,随着勃起的分子生物学机制研究所取得的重大突破和磷酸二酯酶 5 (PDE5 )抑制剂西地那非 (sildenafil,Viagra)的问世 ,植物性中药治疗ED的研究逐渐转向提取有效的中药单体及阐明其作用机制 ,并取得了可喜的成绩。本文综述了近年来ED的中药治疗研究进展     

兔阴茎海绵体平滑肌细胞的体外培养及其生物学特性

目的 :研究体外培养的新西兰白兔阴茎海绵体平滑肌细胞的生物学特性。　方法 :采用组织块培养法 ,对兔阴茎海绵体平滑肌细胞进行活细胞观察 ,并测定其细胞生长曲线、贴壁率、细胞分裂指数。　结果 :①兔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 ;②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。　结论 :体外培养的兔阴茎海绵体平滑肌细胞模型可用于检测某些药物对阴茎勃起的影响。     

siRNA敲减整合素连接激酶影响人阴茎海绵体平滑肌细胞功能的体外研究

目的 探讨整合素连接激酶( ILK)基因对人海绵体平滑肌细胞收缩和舒张功能的影响.方法 人海绵体平滑肌细胞为实验对象,分为非沉默组、非处理对照组和ILK小干扰RNA(siRNA)实验组,采用siRNA干扰技术下调细胞中ILK的表达,伸展实验和基质胶黏附实验分别检测ILK基因敲减前后海绵体平滑肌细胞伸展和黏附能力变化,Transwell小室观察ILK表达对海绵体平滑肌细胞迁移能力的影响,荧光标记的鬼笔环肽染色海绵体平滑肌细胞观察ILK基因敲减前后细胞 骨架的变化.结果 黏附实验中,非沉默siRNA组、非处理对照组和实验组siRNA干扰的平滑肌细胞吸光度(A)值分别为0.184±0.034、0.179±0.028和0.092±0.010;迁移实验中,3组穿过Matrigel胶包被的Transwell小室的平滑肌细胞A值分别为0.184±0.017、0.188 ±0.013和0.106±0.003,实验组与非沉默siRNA组比较差异有统计学意义(P＜0.05);非处理对照组平滑肌细胞应力纤维错综分布于细胞周边与胞质内,而实验组的细胞应力纤维仅分布于细胞边缘.结论 ILK下调降低人海绵体平滑肌细胞的黏附、伸展、迁移能力,并通过影响细胞骨架重构参与调节平滑肌收缩与舒张状态,提示ILK基因有可能成为勃起功能障碍基因治疗的有效靶点.     

血管性勃起功能障碍动物模型的建立

目的探索建立血管性勃起功能障碍(ED)动物模型。方法雄性新西兰大白兔给予胆固醇及高脂饮食,同时用球囊导管损伤双侧髂内动脉内皮。测定术后4周、8周血脂并观察阴茎勃起情况、交配情况,同时用阴茎海绵体注射盐酸罂粟碱、阴茎血管双核素检查、髂内动脉数字减影造影(DSA)等方法研究勃起功能。结果与对照组相比,术后4周、8周模型组动物出现明显高脂血症,勃起程度明显下降,交配实验的各项观察指标均显著降低(P＜0.01),动脉造影显示双侧髂内动脉管腔直径明显缩小,双核素检查动脉显像指数(PIA)及静脉显像指数(PIV)明显降低,但第4周的模型组与对照组PIV结果没有统计学差异(P＞0.05)。结论高脂、高胆固醇饮食同时用球囊导管扩张动物髂内动脉,可以加速模型的形成,从而用于血管性ED的进一步研究。     

降钙素基因相关肽对糖尿病性ED大鼠阴茎海绵体平滑肌细胞表型转化的影响

目的:了解降钙素基因相关肽(CGRP)对糖尿病性ED大鼠阴茎海绵体平滑肌细胞表型转化的影响。方法:利用链脲佐菌素建立糖尿病及糖尿病性ED大鼠模型。阴茎海绵体平滑肌细胞原代培养,并进行免疫细胞化学染色鉴定。实验分为2组:正常对照组和糖尿病性ED大鼠组。不同浓度(0、10,60,100 nmol/L)CGRP作用24h后,利用qRT-PCR检测各组细胞碱性调宁蛋白和骨桥蛋白mRNA的表达。结果:各组原代培养阴茎海绵体平滑肌细胞α-肌动蛋白阳性细胞率为(95.94±0.03)%。与正常对照组比较,糖尿病组ED大鼠阴茎海绵体平滑肌细胞碱性调宁蛋白mRNA表达显著减少(4.41±0.29 vs 10.35±0.62,P<0.01),而骨桥蛋白mRNA表达水平显著上调(5.28±0.32 vs 1.32±0.24,P<0.01)。当CGRP作用的终浓度为100 nmol/L时,糖尿病组大鼠阴茎海绵体平滑肌细胞经CGRP作用后,与未经其作用相比,碱性调宁蛋白mRNA表达显著上调(6.90±0.22 vs 4.41±0.29,P<0.01),而骨桥蛋白mRNA表达水平显著减少(3.26±0.31 vs 5.28±0.32,P<0.01)。结论:CGRP可使糖尿病性ED大鼠阴茎海绵体平滑肌细胞表型从合成型向收缩型转化。     

Rehabilitation of the cavernous smooth muscle in patients with organic erectile dysfunction

This study aimed at assessing the effect of regular use of intracorporeal injection (ICI), sildenafil citrate and vacuum constriction device (VCD) on cavernous smooth muscle and erectile activity. One hundred and sixty-five patients with organic erectile dysfunction were investigated for 3 months. The patient and his partner were classified prospectively after proper counselling: group I (n = 56) received ICI twice per week; group II (n = 55) received sildenafil 100 mg twice per week; and group III (n = 54) used VCD twice per week. Duplex ultrasound was carried out before and after treatment, and then, the patients were followed up for a month to assess the resumption of unaided erection. The results showed that there was significant improvement in mean peak systolic velocity (PSV) and mean cavernosal artery diameter (CAD) at the end of the treatment in all groups, being higher in the ICI group than in the other two groups. Also, the percentage of patients who resumed unaided intercourse were higher in the ICI group compared with the other two groups (17.9%, 9.1% and 3.7% respectively). It is concluded that repeated regular use of ICI, sildenafil or VCD by patients with organic erectile dysfunction has a positive impact on their cavernous blood flow and erectile activity.     

Endovascular treatment of vasculogenic erectile dysfunction

The treatment of erectile dysfunction (ED) has been a fascination involving multiple medical specialities over the past century with urologic, cardiac and surgical experts all contributing knowledge toward this multifactorial disease. With the well-described association between ED and cardiovascular disease, angiography has been utilized to identify vasculogenic impotence. Given the success of endovascular drug-eluting stent (DES) placement for the treatment of coronary artery disease, there has been interest in using this same technology for the treatment of vasculogenic ED. For men with inflow stenosis, DES placement to bypass arterial lesions has recently been reported with a high technical success rate. Comparatively, endovascular embolization as an approach to correct veno-occlusive dysfunction has produced astonishing procedural success rates as well. However, after a thorough literature review, arterial intervention is only recommended for younger patients with isolated vascular injuries, typically from previous traumatic experiences. Short-term functional outcomes are less than optimal with long-term results yet to be determined. In conclusion, the hope for a minimally invasive approach to ED persists but additional investigation is required prior to universal endorsement.     

内皮细胞凋亡与勃起功能障碍的研究进展

勃起功能障碍(ED)是泌尿男科最常见的疾病之一,严重影响患者的生活质量,引起ED的因素包括衰老、糖尿病、高血压、高脂血症以及不良生活方式等,其确切机制尚未完全明确。目前随着对ED研究的不断深入,其中阴茎海绵体内皮细胞凋亡与ED的关系倍受关注,内皮细胞凋亡增加可引起NOS活性降低导致NO合成受阻,从而影响阴茎勃起,不同病因引起的ED其内皮细胞凋亡的机制不同。本文就阴茎海绵体内皮细胞凋亡在ED中的作用作一综述,对ED与内皮细胞凋亡机制的深入了解将为新药研制及其他治疗措施提供新的思路。     

阴茎海绵体平滑肌细胞外钙离子浓度在体检测与神经性ED的相关研究

目的 通过钙离子传感针在体检测阴茎海绵体平滑肌细胞外的钙离子浓度,探讨其与勃起功能障碍(ED)的相关性.方法 40只雄性SD大鼠随机分为损伤组(20只)和对照组(20只),通过剪断双侧海绵体神经制作神经性ED模型,1个月后应用钙离子传感针在体检测大鼠阴茎海绵体平滑肌细胞外的钙离子浓度,比较两组间钙离子浓度的差异.结果 损伤组(ED组)钙离子浓度(电位)为(-0.31±0.35)mV,对照组钙离子浓度(电位)为(0.17±0.34)mV,两组钙离子浓度差异具统计学意义(P<0.05).结论 钙离子传感针可以作为ED在体检测的一种新方法.     

Myocardin restores erectile function in diabetic rats: phenotypic modulation of corpus cavernosum smooth muscle cells

This study aimed to investigate whether gene transfer of myocardin to the penis of diabetic rats can modulate corpus cavernosum smooth muscle (CCSM) cells phenotype and restore erectile function. Five normal control rats, and 22 diabetic rats were randomly divided into four groups: rats transfected with adCMV‐myocardin (N = 6), treated with empty vector (N = 6), injected with medium (N = 5), and sham‐operated rats (N = 5). The erectile response was measured 7 days after transfection. The percent of smooth muscle and the expressions of SMα‐actin, smooth muscle myosin heavy chain (SMMHC), calponin were evaluated. The increases in intracorporal pressure(ICP)/mean arterial pressure and total ICP in response to nerve stimulation in the adCMV‐myocardin treated rats were significantly greater than those in the empty vector (P < 0.001 and P < 0.001), medium only (P < 0.001 and P < 0.001), and sham‐operated rats (P < 0.001 and P < 0.001). The suppressed expressions of SMα‐actin, SMMHC and calponin were completely restored, and the amount of smooth muscle in diabetic rats were not restored after treatment. It is concluded that myocardin ameliorated erectile responses in diabetic rats mainly via promoting phenotypic modulation of CCSM cells from a proliferative to a contractile state.     

粉防己碱(Tet)对兔子阴茎海绵体平滑肌细胞质中自由钙离子浓度的影响(英文)

目的:探讨粉防己碱(tetrandrine,Tet)松弛阴茎海绵体平滑肌的作用机制。方法:体外培养新西兰白兔阴茎海绵体平滑肌细胞,经钙荧光指示剂 Fluo-2/AM 负载后,用荧光离子数字成像系统观察 Tet 对平滑肌细胞内[Ca~(2 )]_i 的影响。结果:Tet(1,10,100μmol/L)对平滑肌细胞内静息[Ca~(2 )]_i无明显影响(P>0.05)。当细胞外钙离子浓度为2.5 mmol/L 时,Tet(1μmol/L,10 μmol/L,100 μmol/L)抑制了高钾和去氧肾上腺素(PE)导致的细胞内[Ca~(2 )]_i 升高(P<0.05),这种抑制作用具有浓度依赖性。在无细胞外钙时,1 μmol/L和10μmol/L Tet 对 PE 引起的细胞内[Ca~(2 )]_i 升高无明显影响(P>0.05);而100 μmol/L Tet 能明显抑制 PE 引起的细胞内[Ca~(2 )]_i 升高(P<0.05)。结论:Tet 通过阻滞电压依赖性钙通道、α_1受体依赖性钙通道和抑制细胞内钙库释放,降低阴茎海绵体平滑肌细胞内[Ca~(2 )]_i 水平,这是 Tet 松弛阴茎海绵体平滑肌的作用机制之一。     

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.