机械牵张对豚鼠膀胱组织Cajal间质细胞形态学及兴奋性的影响

目的 探讨机械牵张对豚鼠膀胱组织Cajal间质细胞(ICC)形态及兴奋性的影响.方法 建立雌性豚鼠膀胱颈部分梗阻(PBOO)模型作为实验组.并设假手术组作为对照.术后4周取膀胱组织制片,免疫荧光染色观察2组ICC形态和分布情况;胶原酶消化豚鼠膀胱制细胞悬液,免疫荧光标记,流式细胞仪检测2组c-kit阳性细胞比率.在弹性硅胶膜七原代培养膀胱ICC,利用Fluo-4钙荧光指示剂检测机械牵张对ICC钙信号的影响.结果 2组豚鼠膀胱组织铺片均可见c-kit染色阳性典型长梭形有突起的ICC,主要分布于平滑肌肌束间;PBOO组膀胱平滑肌间质增厚,c-kit染色阳性细胞数量及其突起明显增多,互相连接呈网络状;PBOO组c-kit阳性细胞比率为(6.7±1.7)%,显著高于对照组的(1.0±0.5)%,差异有统计学意义(P＜0.05).体外培养的ICC可检测到自发性钙波,机械牵张刺激可诱导ICC发生钙波增强现象.结论 机械牵张可诱导膀胱ICC兴奋性增强;PBOO膀胱组织中ICC数量及相互联系显著增多.ICC可能参与了膀胱牵张感受功能并在长期牵张应力负荷下发生一定的适应性改变.     

小鼠胆囊酪氨酸激酶受体的表达及其意义

目的研究酪氨酸激酶受体（c-kit）在CD1小鼠胆囊的表达及其意义。方法采用免疫组化方法，应用激光扫描共聚焦显微镜检测CD1小鼠胆囊组织切片和急性分离单细胞的c-kit免疫反应阳性细胞的表达；RT-PCR法检测胆囊c-kit mRNA的表达；Western blot检测胆囊c-kit蛋白的表达。结果免疫荧光激光扫描共聚焦显微镜检测结果显示CD1小鼠胆囊从黏膜层到肌层均有c-kit免疫反应阳性细胞存在，其呈类似网络状分布和点和（或）丝状分布。急性分离胆囊单细胞检测结果显示c-kit免疫反应阳性细胞大致可区分为两种类型，一种为分支状，大多数细胞呈现该形态；一种为类梭形，少见分支，类似平滑肌细胞，部分细胞呈现此形态。而典型呈梭形的平滑肌细胞则无荧光显现，呈c-kit免疫反应阴性。RT-PCR法检测结果表明胆囊c-kit mRNA表达阳性。Western印迹显示c-kit蛋白表达阳性。结论c-kit在CD1小鼠胆囊表达阳性，即CD1小鼠胆囊存在Cajal间质细胞（ICC）。ICC可能与胆囊运动有关，有可能为胆囊起搏细胞。     

人体阴茎海绵体神经一氧化氮合成酶免疫组织化学研究

目的探讨人体阴茎海绵体神经中一氧化氮合成酶的分布情况。方法采用人体组织切片苏木素-伊红(HE)染色和神经性一氧化氮合成酶(nNOS)免疫组织化学染色方法，对1具成人尸体阴茎海绵体神经及相关组织进行染色分析。结果前列腺后外侧神经束中存在nNOS神经元细胞体和神经纤维。神经元细胞胞浆内可见均匀分布棕黄色颗粒，神经纤维则可见散在棕黄色颗粒。阴茎干背神经中，4条神经纤维截面发现有棕黄色颗粒，其他神经纤维均未发现棕黄色颗粒。龟头部组织中，未发现nNOS棕黄色颗粒神经纤维。结论前列腺后外侧阴茎海绵体神经束中存在nNOS，并且包含nNOS神经节细胞，它发出nNOS神经纤维到达阴茎海绵体，与背神经同行。     

豚鼠膀胱组织内Cajal样间质细胞及NOS表达阳性神经元的分布

目的探讨Cajal样间质细胞和一氧化氮合酶2（NOS2）表达阳性神经元在成年豚鼠膀胱组织内的分布。方法成年豚鼠膀胱冰冻切片,c—Kit免疫荧光和n—NOS2免疫荧光染色。结果膀胱肌层内可见c—Kit表达阳性细胞的存在,n-NOS2免疫荧光染色显示其阳性神经元广泛分布于膀胱组织内。c—Kit免疫荧光和n-NOS2免疫荧光双重染色虽未发现两者的共存,但可见c—Kit表达阳性细胞存在于NOS表达阳性神经元的附近。结论豚鼠膀胱的Cajal样间质细胞可能参与膀胱自主节律性运动的调控,提示二者可能共同参与膀胱自主节律性运动的调控,为“神经-Cajal样问质细胞-肌肉”作用单元的形成提供结构基础。     

大鼠膀胱ICC样细胞与逼尿肌神经调控关系的形态学研究

目的 从形态学上探讨膀胱ICC样细胞在逼尿肌神经调控中的作用.方法 采用透射电镜观察大鼠膀胱内ICC样细胞、神经和逼尿肌细胞之间的超微结构关系.通过c-kit免疫荧光染色对大鼠膀胱ICC样细胞进行鉴定,并通过c-kit与PGP9.5免疫荧光双标观察ICC样细胞与神经的结构关系.结果 透射电镜显示,ICC样细胞与逼尿肌细胞紧密相邻处可见典型缝隙连接.在局部区域可见ICC样细胞的突起与神经末梢联系紧密.c-kit免疫荧光染色显示,大鼠膀胱内ICC样细胞主要位于黏膜下层、肌束边缘以及肌细胞间.c-kit与PGP9.5免疫荧光双标显示ICC样细胞与神经末梢在结构上关系紧密.结论 从形态学上看,膀胱内ICC样细胞具备参与逼尿肌神经调控的结构基础,进一步从功能学上予以证实将有助于全面阐明逼尿肌神经调控理论.     

骨髓间充质干细胞即刻与延时治疗修复阴茎海绵体神经损伤大鼠勃起功能的研究

目的:拟观察在双侧阴茎海绵体神经(CN)损伤的大鼠神经性勃起功能障碍(NED)模型中即刻和延时向阴茎海绵体注射骨髓间充质干细胞(BM-MSCs)对大鼠阴茎勃起功能的修复作用。方法:选取28只8周龄、体重200~250 g雄性SD大鼠随机分为4组:假手术组找到双侧海绵体神经后不做处理直接关腹并缝合皮肤;对照组、即刻治疗组和延时治疗组均通过钳夹的方式损伤双侧阴茎海绵体神经建立NED模型随后关腹缝合。假手术组和对照组向阴茎海绵体内注射对照剂,即刻治疗组注射BM-MSCs,延时治疗组术后两周注射BM-MSCs。术后12周采用电刺激CN记录阴茎海绵体内压(ICP),颈动脉穿刺测定平均动脉压(MAP),ICP/MAP作为勃起功能的评价指标来评估大鼠的勃起功能。在勃起功能检测后处死大鼠,取阴茎海绵体中段组织检测平滑肌、胶原和神经纤维。结果:在手术后12周,即刻治疗组和延时治疗组ICP和ICP/MAP均较对照组升高(P0.05)。即刻治疗组和延时治疗能提高大鼠海绵体组织内平滑肌与胶原的比例(P0.05),同时两组阴茎海绵体内平滑肌含量高于对照组(P0.05),阴茎海绵体背神经内神经微丝(NF)蛋白阳性神经纤维数目和nNOS的表达高于对照组(P0.05)。结论:阴茎海绵体内注射BM-MSCs能对双侧CN损伤大鼠的勃起功能恢复有促进作用。BM-MSCs治疗可以提高海绵体组织内平滑肌与胶原的比例,改善纤维化,并能提高海绵体组织中平滑肌含量、阴茎背神经的神经丝含量和nNOS的表达水平,术后延时治疗同样能取得一定的治疗效果。     

FK506对大鼠阴茎海绵体神经再生影响的研究

目的:探讨FK506对大鼠阴茎海绵体神经损伤后再生的影响,并讨论其作用的可能机制。　方法:54只 SD雄性大鼠随机分成3组,即假手术对照组(简称对照组,n=24)、单侧阴茎海绵体神经切断组(简称单切组,n= 24)、单侧阴茎海绵体神经切断+FK506组(简称FK506组,n=6)。术后1、3个月电刺激阴茎海绵体神经并连续监 测阴茎海绵体内压变化及阴茎勃起情况,同时取阴茎海绵体组织采用NADPH d法观察一氧化氮合酶(nNOS)阳性 神经纤维的再生情况。　结果:术后1个月单切组nNOS阳性神经纤维数量明显减少,与对照组相比差异有极显 著性(P<0.01),术后3个月在电刺激未切断侧阴茎海绵体神经时,FK506组比单切组产生更大的最大海绵体内压 (P<0.01),且单切组阴茎海绵体组织中nNOS阳性神经纤维比术后1个月无明显增加(P>0.05),而FK506组 nNOS阳性神经纤维显著增加(P<0.01)。　结论:FK506皮下注射能促进大鼠损伤的阴茎海绵体神经再生,促进 勃起功能恢复。     

豚鼠Oddi括约肌内Cajal样细胞及NOS表达阳性神经元的分布

目的研究Cajal样细胞和一氧化氮合酶(nitric oxide synthase,NOS)表达阳性神经元在成年豚鼠Oddi括约肌(sphincter of Oddi,SO)的分布。方法成年豚鼠SO冷冻切片,c-Kit免疫细胞化学和NADPH-黄递酶组织化学双重染色。结果SO横切面环行平滑肌层内可见少量c-Kit阳性细胞,胞体呈梭形,两端伸出细长的突起。纵切面SO的两个壁分别为:外侧壁和十二指肠壁内SO壁,前者与肠壁结构类似,在深肌丛和肌间神经丛周围可见较多的c-Kit阳性细胞;而十二指肠壁内SO壁的肌层内存在大量c-Kit阳性细胞和突起,其形态与肠壁肌层内Cajal细胞相似。NADPH-黄递酶组织化学染色可见NOS表达阳性神经元广泛分布于SO的肌间神经丛和平滑肌内。c-Kit免疫细胞化学和NADPH-黄递酶组织化学双重染色虽未发现二者的共存,但可见c-Kit阳性细胞及突起存在于NOS表达阳性神经元的附近。结论豚鼠SO内存在的Cajal样细胞可能参与SO自主节律性运动的调控,可能是NOS表达阳性神经元对SO运动发挥调节作用的靶细胞。     

神经源性膀胱组织中一氧化氮合酶表达的特点和意义

目的 探讨神经元型一氧化氮合酶(nNOS)、诱导型NOS(iNOS)和内皮型NOS(eNOS)在神经源性膀胱组织中表达状况,探讨一氧化氮在神经源性膀胱组织中产生及作用特点.方法 神经源性膀胱患儿30例.男18例,女12例.年龄(6.3±3.1)岁.30例均行手术治疗,术中留取膀胱组织标本,采用免疫组织化学方法检测膀胱组织中nNOS、iNOS和eNOS表达状况,10例正常膀胱组织标本作对照. 结果 正常膀胱体部组织nNOS阳性表达,走行在平滑肌纤维之间,分布于平滑肌细胞表面,膀胱基质也有表达,组织化学评分(HS)为2.8～4.0和1.2～2.7;平滑肌细胞iNOS阴性表达,平滑肌细胞基质有少量稀疏表达,HS为0～0.4和0～0.1;eNOS表达分布于血管内皮细胞中,分布稀疏,平滑肌细胞无表达.膀胱颈部组织表达高于膀胱体部组织,以nNOS表达为主.神经源性膀胱组织中以iNOS表达为主,nNOS表达明显减少;eNOS主要分布于膀胱基质的内皮细胞,膀胱平滑肌、成纤维细胞阴性表达;病变膀胱组织血管稀疏,微血管密度100倍视野下可见到(6.8±3.2)个血管灶,低于正常膀胱组织的(16.7±6.3)个(P＜0.01).结论 正常膀胱组织nNOS主要分布在膀胱颈中,NO合成主要受nNOS调节.神经源性膀胱患者膀胱组织中氮能神经元分布稀疏,nNOS表达减少,iNOS表达上调,NO的合成与调节可能主要来源于iNOS,受iNOS水平调节,eNOS表达下降,提示膀胱组织血供不良.     

生长激素补充对老龄大鼠勃起功能及阴茎海绵体nNOS表达的影响

目的探讨生长激素(GH)补充对老龄大鼠勃起功能及阴茎海绵体神经型一氧化氮合酶(nNOS)表达的影响。方法20只18月龄SD大鼠随机均分成A、B两组,10只2月龄SD大鼠为C组。A组给予GH1U/(kg·d),B、C组给予相同剂量的生理盐水,均皮下注射8周。于8周末观察阿朴吗啡(APO)皮下注射与大鼠海绵体内注射罂粟碱诱导的阴茎勃起情况,并用免疫组化SP法检测阴茎海绵体组织中nNOS神经纤维的数目。结果8周末时,A、C组大鼠APO诱导的勃起次数、海绵体内注射罂粟碱诱导的最大海绵体内压以及阴茎海绵体组织中nNOS神经纤维数目均显著高于B组(P<0.05或P<0.01)。结论老龄大鼠勃起功能及阴茎海绵体nNOS表达较成年大鼠明显下降,GH补充可以部分改善老龄大鼠的勃起功能,其机制之一可能与GH补充增加了老龄大鼠阴茎海绵体组织中nNOS神经纤维数目有关。     

Ultrastructural features and possible functional role of kit-positive interstitial cells in the guinea pig corpus cavernosum

The objective of the present study was to identify kit-positive interstitial cells (ICs) in guinea pig corpus cavernosum and examine their relationships with adjacent smooth muscle cells (SMCs) and intramural nerves. In addition, we investigated the possible involvement of ICs in nitric oxide (NO)-mediated relaxation of corpus cavernosum smooth muscle (CCSM). ICs were identified by their immunoreactivity to the kit receptor, a cell surface marker encoded by c-kit proto-oncogene and specific for interstitial cells of Cajal. ICs were abundantly distributed in guinea pig corporal tissues. Ultrastructural investigation by conventional transmission electron microscopy revealed the ultrastructural features of ICs and gap junctions located between ICs and adjacent SMCs, furthermore, a close contact between ICs and intramural nerves for the first time. Western blot analysis of purified ICs by fluorescence-activated cell sorting revealed coexpression of soluble guanylate cyclase (sGC)α1, sGCβ1 and kit receptor tyrosine kinase protein in them. These observations imply that ICs express the NO-sensitive sGC molecule and may be involved in the NO-mediated relaxation of CCSM in the guinea pig corpus cavernosum.     

Kit positive cells in the guinea pig bladder

PURPOSE: We describe the presence of interstitial cells of Cajal (ICC) throughout the wall of the guinea pig bladder. MATERIALS AND METHODS: Bladders obtained from male guinea pigs were prepared for immunohistochemical investigations using various primary antibodies, including the specific ICC marker c-kit (Gibco BRL, Grand Island, New York). Enzymatically dispersed cells with a branched morphology were identified as ICC using anti-c-kit. They were loaded with fluo-4acetoxymethyl (Molecular Probes, Eugene, Oregon) and studied using confocal laser scanning microscopy. RESULTS: Anti-c-kit labeling demonstrated that ICC were oriented in parallel with the smooth muscle bundles that run diagonally throughout the bladder. Double labeling with anti-smooth muscle myosin (Sigma Chemical Co., St. Louis, Missouri) revealed that ICC were located on the boundary of smooth muscle bundles. When anti-c-kit was used in combination with the general neuronal antibody protein gene product 9.5 (Ultraclone Ltd., Isle of Wight, United Kingdom) or anti-neuronal nitric oxide synthase, it was noted that there was a close association between nerves and ICC. Enzymatic dissociation of cells from tissue pieces yielded a heterogeneous population of cells containing typical spindle-shaped smooth muscle cells and branched cells resembling ICC from other preparations. The latter could be identified immunohistochemically as ICC using anti-c-kit, whereas the majority of spindle-shaped cells were not Kit positive. Branched cells responded to the application of carbachol by firing Ca2+ waves and they were often spontaneously active. CONCLUSIONS: ICC are located on the boundary of smooth muscle bundles in the guinea pig bladder. They fire Ca2+ waves in response to cholinergic stimulation and can be spontaneously active, suggesting that they could act as pacemakers or intermediaries in the transmission of nerve signals to smooth muscle cells.     

Effect of diabetes and insulin treatment on nitric oxide synthase content in rat corpus cavernosum

AIM: To study the effect of diabetes mellitus and insulin treatment on rat penile nitric oxide synthase content. METHODS: Male Wistar rats were divided at random into two groups: the Control (n = 8) and the Diabetic (n = 17). Diabetes mellitus was induced by intraperitoneal injection of streptozotocin. The diabetic animals were then randomly divided into two subgroups: diabetic rats without insulin treatment (n = 7) and diabetic rats with insulin treatment (n = 10). The neuronal nitric oxide synthase (nNOS) in the penile corpus cavernosum were assayed by immumohistochemical staining with specific antibody to nNOS and the nNOS-positive nerve fibers were counted semiquantitatively under a high power microscope. RESULTS: The nNOS- positive nerve fibres in diabetic rats with treatment was higher than that in diabetic rats without treatment (P < 0.05) and lower than that in the controls (P < 0. 01). The nNOS-positive nerve fibres in diabetic rat without treatment were also lower than that in the controls (P < 0. 01). CONCLUSION: In streptozotocin-induced diabetic rats, the nNOS content in the penile corpus cavernosum was significantly decreased. Insulin treatment at the dose level employed partially restores the penile nNOS content in these rats.     

Effect of diabetes and insulin treatment on nitric oxide synthase content in rat corpus cavemosum

Aim: To study the effect of diabetes mellitus and insulin treatment on rat penile nitric oxide synthase content.Methods: Male Wistar rats were divided at random into two groups: the Control (n = 8) and the Diabetic (n =17). Diabetes mellitus was induced by intraperitoneal injection of streptozotocin. The diabetic animals were then ran-domly divided into two subgroups; diabetic rats without insulin treatment (n = 7) and diabetic rats with insulin treat-ment (n = 10). The neuronal nitric oxide synthase (nNOS) in the penile corpus cavernosum were assayed by immumo-histochemical staining with specific antibody to nNOS and the nNOS-positive nerve fibers were counted semiquantita-tively under a high power microscope. Results: The nNOS- positive nerve fibres in diabetic rats with treatment washigher than that in diabetic rats without treatment ( P < 0.05) and lower than that in the controls ( P < 0.01). ThenNOS-positive nerve fibres in diabetic rat without treatment were also lower than that in the controls (     

Immunolocalization of the gap junction protein Connexin43 in the interstitial cells of Cajal in the normal and Hirschsprung's disease bowel

BACKGROUND: Interstitial cells of Cajal (ICC) are pacemaker cells between gastrointestinal smooth muscles; they generate spontaneous slow waves of the smooth muscle layers and mediate neurotransmission. The cellular network of ICC is connected by Gap junctions to each other and to the smooth muscle cells. Although there have been several studies reporting distribution of ICC in the normal bowel and pathological conditions such as Hirschsprung's disease, there is little information on the crucial role of Gap junctions in the intercellular communication in the gut musculature. The aim of this study was to investigate the immunolocalization of the Gap junction protein Connexin43 in the normal and Hirschsprung's disease (HD) bowel using whole-mount preparation technique and confocal laser scanning microscopy. METHODS: Full-thickness bowel specimens were collected at pull-through operation from 8 patients diagnosed as having HD. Normal control large bowel specimens were collected from 12 patients during bladder augmentation operation. Whole-mount preparation was performed on all specimens and double immunostaining was carried out using anti c-kit and antiConnexin43 antibodies. The immunolocalization was detected with the help of confocal laser scanning microscopy. RESULTS: Connexin43 immunoreactivity appeared in and between the c-kit-positive cells and along the smooth muscle fibers of the normal bowel and ganglionic part of HD bowel. In the aganglionic part of HD bowel there was no expression of Connexin43. In the transitional zone of HD the Connexin43 staining was weak and colocalized only in the processes of the c-kit-positive Cajal cells. CONCLUSIONS: Results of this study show for the first time that Gap junctional protein Connexin43 is present in the ICCs, which form a 3-dimensional network in the normal bowel wall. The lack of expression of Connexin43 in the aganglionic bowel and reduced expression in the transitional zone of HD suggest that the impaired intercellular communication between ICCs and smooth muscle cells may partly be responsible for the motility dysfunction in HD.     

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     

大鼠小肠Cajal间质细胞的体外分离、培养及鉴定

目的：探讨大鼠小肠Cajal间质细胞（ICC）的原代分离、培养及鉴定方法。 方法：出生后5~10 d的SD乳大鼠,颈椎脱臼处死。无菌条件下取出小肠,在解剖显微镜下剥去小肠系膜、肠黏膜和黏膜下层,将小肠平滑肌层组织剪成小块后接种于含有干细胞因子的DMEM培养基中进行培养。倒置显微镜下连续观察组织块周围细胞的游出情况及细胞的形态,用酪氨酸蛋白激酶受体c-kit特异性抗体免疫荧光染色鉴定细胞类型。 结果：培养1周后,倒置显微镜下可见组织块周围长出细胞,呈梭形、三角形,有多个短突起;随着培养时间的延长,突起细长化并彼此相互连接形成网络。该类细胞c-kit抗体免疫荧光染色呈阳性。 结论：该研究成功建立了一套由大鼠小肠平滑肌组织块原代培养ICC的方法,为ICC的生物学特性及其与胃肠道动力障碍性疾病关系的研究奠定了基础。     

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.