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.     

豚鼠膀胱Cajal样间质细胞类型及其与钙离子振荡特性的关系

目的 探讨豚鼠膀胱组织中Cajal样间质细胞(ICC)形态学类型特点及其与钙离子振荡特征的关系.方法 取豚鼠膀胱制作冰冻切片,体外培养膀胱ICC.免疫荧光染色激光共聚焦显微镜下观察ICC形态学特点,根据细胞不同形态类型及添加2-氨基乙氧基双苯萘酯(2-APB)干预剂将体外培养膀胱ICC分4组:二聚体ICC组(n=10)、单体ICC组(n=20)、2-APB抑制二聚体ICC组(n=10)、2-APB抑制单体ICC组(n=20).应用Fluo-4AM标记细胞内钙离子浓度含量及在2-APB抑制二聚体ICC组、2-APB抑制单体ICC组加入2-APB,共聚焦显微镜下观察记录钙离子振荡功能.结果 二聚体ICC组钙振荡频率(13.4±0.8)次/min及振幅38.5±8.7明显高于单体ICC组钙振荡频率(7.5±0.6)次/min及振幅10.5±3.9(P＜0.05),加入高浓度2-APB后,2-APB抑制二聚体ICC组钙振荡频率(8.7±1.5)次/min及幅度15.5±8.2明显低于二聚体ICC组(P＜0.05),而2-APB抑制单体ICC组钙振荡频率(8.5±0.5)次/min及幅度9.2±4.6与单体ICC组差异无统计学意义(P＞0.05).结果 豚鼠膀胱中存在单体和二聚体2种不同形态类型的ICC,单体ICC自发性钙振荡波较低,二聚体ICC自发性钙振荡波具有高兴奋性,二聚体ICC的特有结构可能是ICC高兴奋性钙波形成的结构基础.     

豚鼠膀胱cajal样细胞的类型

目的：观察豚鼠膀胱cajal样细胞形态学特点并结合该细胞具有的特殊功能,探讨cajal样细胞在膀胱组织中的类型。方法：豚鼠膀胱经手术取材后制成冷冻切片和体外培养膀胱cajal样细胞,采用间接免疫荧光染色后在激光共聚焦显微镜观察cajal样细胞形态学特点,然后将体外培养的膀胱cajal样细胞应用Fluo一4AM标记细胞内钙离子浓度含量,在激光共聚焦显微镜下观察cajal样细胞钙离子振荡功能。结果：膀胱组织冷冻切片和体外cajal样细胞培养均可见单体cajal样细胞和二聚体cajal样细胞,体外培养的两种类型的cajal样细胞具有钙离子振荡功能。结论：豚鼠膀胱组织中存在单体和二聚体两种不同类型的cajal样细胞。     

逼尿肌反射亢进大鼠膀胱ICC细胞兴奋性变化及意义

目的:观察脊髓损伤后大鼠逼尿肌反射亢进膀胱Cajal间质细胞(ICC)细胞兴奋性的变化,探讨膀胱逼尿肌功能改变与ICC细胞兴奋性变化的可能关系。方法:制作大鼠骶髓上损伤模型,膀胱压力测定证实膀胱出现逼尿肌反射亢进后,取膀胱组织急性分离ICC细胞,激光共聚焦显微镜观察ICC细胞兴奋性变化。结果:逼尿肌反射亢进大鼠模型制作成功,膀胱ICC细胞兴奋性较正常组和膀胱造瘘组明显增加。结论:脊髓损伤后大鼠膀胱功能改变可能与ICC细胞兴奋性增加有关。     

草氨酸盐对人胰腺癌细胞内钙含量影响的研究

目的:探讨草氨酸盐(oxamate)对人胰腺癌细胞内钙含量的影响。方法:体外培养人胰腺癌细胞panc-1，用oxamate干预癌细胞后48h，行钙离子荧光指示剂Fluo-3/AM染色，激光共聚焦下观察不同浓度oxamate作用下细胞内钙含量的变化。结果:oxamate干预48h后，细胞内钙含量随着oxamate浓度的升高而致荧光强度逐渐增强，呈剂量-效应关系。结论:oxamate能增加panc-1细胞内钙离子含量，影响细胞内钙离子平衡，进而干涉细胞病理生理过程。     

逼尿肌反射亢进大鼠膀胱ICC细胞数量的变化及意义

目的：观察脊髓损伤后大鼠逼尿肌反射亢进膀胱Cajal间质细胞（ICC）数量的变化,探讨膀胱逼尿肌功能改变和ICC的可能关系。方法：制作大鼠骶髓上损伤模型,膀胱压力测定证实膀胱出现逼尿肌反射亢进后,取膀胱组织行ckit免疫组织化学染色．用激光共聚焦显微镜观察ICC数量的变化。结果：逼尿肌反射亢进大鼠模型制作成功,膀胱ICC数量较正常组和膀胱造瘘组明显增加。结论：脊髓损伤后大鼠膀胱逼尿肌反射亢进可能由ICC数量变化所引起。     

大黄素对体外培养Cajal间质细胞钙离子振荡功能的影响

目的:探讨大黄素对体外培养Cajal间质细胞钙离子振荡的影响,从而进一步揭示大黄素等促进胃肠动力的分子机制方法:体外培养小鼠ICC,体外添加大黄素,应用钙离子指示剂标记细胞内钙离子含量,在激光共聚焦显微镜下动态观察其钙离子振荡功能的变化。结果:免疫荧光鉴定结果证实体外培养细胞为ICC,体外培养ICC具有钙离子振荡功能,大黄素能显著增强其钙离子振荡的频率和振幅。结论:大黄素能显著增强体外培养ICC钙离子振荡功能,可能是其促进胃肠动力的重要机制。     

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.     

光动力疗法对人膀胱癌细胞胞内游离钙的影响

目的:探讨光动力学作用后的膀胱癌细胞(T-24和SCaBER)胞内钙离子浓度的变化.方法:采用MTT比色分析法判断光动力学对体外人膀胱癌T-24和SCaBER细胞的杀伤效应.同时应用激光共聚焦显微镜和Fluo-3/AM探针技术测定光动力学作用后细胞内钙离子的浓度.结果:T-24和SCaBER细胞内钙离子有明显升高,与对照组比较差异有显著意义(P＜0.001).结论:光动力学作用后细胞内钙离子超载可能在细胞死亡中发挥重要作用.     

肾上腺素β3受体对大鼠逼尿肌细胞钙离子内流的影响

肾上腺素β3 受体( β3 AR)是调节逼尿肌舒张的主要因素[1,2 ] 。我们以Flμo 3AM钙荧光探针检测逼尿肌细胞内游离Ca2 浓度( [Ca2 ]i) ,利用激光共聚焦显微镜技术观察β3 AR激动剂BRL3 73 44A对培养大鼠逼尿肌细胞内Ca2 浓度的影响,并探讨其机制。一、材料与方法1.材料:Wistar大鼠,体重10 0g左右,由第三军医大学实验动物中心提供。Flμo 3AM (购自美国Molecularprobes公司)。2 .逼尿肌细胞原代培养:取体重10 0g左右大鼠,断头处死,摘取膀胱,剪碎使成1mm3 左右碎块,加入1～2ml 0 .1%Ⅳ型胶原酶,3 7℃培养4h ,吹打后将悬液移入离…     

The functional effects of a c-kit tyrosine inhibitor on guinea-pig and human detrusor

OBJECTIVE: To describe the effect of a specific c-kit receptor inhibitor (imatinib mesylate) on human detrusor strips in vitro and guinea-pig cystometry in vivo, and to show histological data suggesting differences in the distribution of interstitial cells of Cajal (ICC)-like cells in 'normal' and overactive human detrusor, as these cells have been identified as possible mediators of spontaneous activity and excitability in bladder smooth muscle. MATERIALS AND METHODS: Specimens of human detrusor were stained immunohistochemically with a c-kit antibody. Human detrusor strips were mounted in a superfused organ-bath apparatus, and smooth muscle contraction was evoked with carbachol and electrical field stimulation in the presence and absence of imatinib mesylate. Also, guinea-pig urodynamic studies were conducted before and after i.v. administration of imatinib mesylate, and changes in bladder variables and spontaneous activity were recorded. RESULTS: Imatinib mesylate (10(-6)M) inhibited evoked smooth muscle contraction and spontaneous activity in overactive human detrusor, with less effect on normal human tissue. Imatinib mesylate (10(-5)M) improved bladder capacity, compliance, voided volumes, urinary frequency, and reduced contraction thresholds and spontaneous activity during guinea-pig cystometry. c-kit labelling showed significantly more ICC-like cells in overactive human detrusor than in normal specimens. CONCLUSION: c-kit receptor blockers have inhibitory effects on guinea-pig and overactive human detrusor, possibly via c-kit receptors on bladder ICC-like cells. This and the possibility that there are more ICC-like cells in overactive bladder suggest that the c-kit receptor may provide a novel target for treating detrusor overactivity.     

银杏内酯B对缺氧环境下大鼠血管平滑肌细胞线粒体钙离子浓度的影响

目的：探讨银杏内酯B对缺氧环境下大鼠血管平滑肌细胞线粒体内钙离子浓度的调节机制。方法：体外培养大鼠血管平滑肌细胞,分为正常对照组（细胞正常培养）、缺氧处理组（细胞在氮气中培养12h）和1、5、10μmol／L银杏内酯B保护组（细胞加入不同浓度的银杏内酯B后在氮气中培养12h）。细胞经荧光探针Rhod-2／AM染色,激光共聚焦扫描显微镜下观察,并计算各组面积积分吸光度值,以此反映线粒体浓度的变化。结果：与正常对照组比较,缺氧造成大鼠血管平滑肌细胞线粒体钙离子浓度显著升高,各银杏内酯B保护组细胞线粒体钙离子浓度明显低于缺氧处理组,且随着银杏内酯B浓度的升高,线粒体钙离子浓度呈下降趋势。结论：银杏内酯B可以维持线粒体内钙离子浓度的稳定,降低缺氧对细胞造成的损伤。     

白细胞介素-37在人外周血CD4＋CD25＋调节性T细胞中的表达

目的：观察白细胞介素-37（IL-37）在健康人外周血 CD4＋CD25＋调节性 T细胞（Treg）中的表达情况。方法：采用免疫磁性分离法分离人外周血 CD4＋CD25＋Treg,流式细胞技术分析其分离纯度,观察刺激剂组（每1×105个 CD4＋CD25＋Treg细胞加入20μl Dynabeads（R） Human Treg Expander）、非刺激剂组、空白对照组（单纯10%FCS-RPMI 1640培养）CD4＋CD25＋Treg细胞增殖活性及其差异。分别采用 Western blot和免疫荧光共聚焦技术检测 IL-37的表达水平与定位改变。结果：MACS分离人外周血 CD4＋CD25＋Treg 细胞纯度为93%,台盼蓝染色细胞活性为98%。与非刺激剂组和空白对照组相比,刺激剂组 CD4＋CD25＋Treg 增殖活性随时间延长而逐渐增加;非刺激剂组与空白对照组其增殖活性差异无显著性。Western blot 结果显示,正常CD4＋CD25＋Treg细胞表达 IL-37,在刺激剂作用下随作用时间延长 IL-37表达量增加。免疫荧光显微镜和免疫荧光共聚焦图像显示,刺激72 h后 IL-37在 CD4＋CD25＋Treg细胞内明显表达,并且在细胞质中表达丰富,近胞膜处表达密度较高。结论：IL-37在健康人外周血 CD4＋CD25＋Treg 细胞中表达,当受到有效刺激后 IL-37表达明显上升。     

慢性非细菌性前列腺炎SD大鼠前列腺平滑肌细胞内钙离子浓度的变化

目的:探讨SD大鼠慢性非细菌性前列腺炎(CAP)炎症组与正常对照组前列腺平滑肌细胞(PSMCs)内游离钙离子浓度([Ca2+]i)的差异及其在CAP中的意义。方法:利用SD大鼠前列腺蛋白提取液与弗氏完全佐剂(FCA)诱导出CAP模型,体外贴壁培养并纯化炎症组与正常对照组PSMCs,细胞经钙离子荧光探针FLUO-3AM孵育后在激光共聚焦显微镜(LSCM)下连续动态扫描。结果:CAP组与对照组的PSMCs钙离子荧光强度分别为80.39±9.00和27.95±10.04,差异具有显著性(P<0.01)。结论:CAP SD大鼠PSMCs[Ca2+]i明显升高,其升高可能增强了PSMCs的收缩作用,从而引起CAP的一系列如对疼痛敏感性增强、下腹部疼痛不适的症状。     

绿色荧光蛋白标记的犬脂肪干细胞体外复合珊瑚材料的生长特性

目的研究犬脂肪干细胞(AdiposeDividedStromalCells,ADSCs)作为种子细复合珊瑚材料体外培养的生长特性。方法分离犬ADSCs,成骨诱导并转染绿色荧光蛋白(GFP)后,复合珊瑚材料培养。检测细胞总数,激光共聚焦观察细胞形态。结果ADSCs-珊瑚复合物体外增殖良好。激光共聚焦显示培养2周后细胞呈老化形态。结论GFP转染不影响细胞传代、增殖。细胞-材料复合物体外培养7天后回植较为适宜。     

CHARACTERIZATION OF CULTURED BLADDER SMOOTH MUSCLE CELLS: ASSESSMENT OF IN VITRO CONTRACTILITY

PURPOSE: The contractile properties of in vitro cultured bladder smooth muscle cells (SMC) are unknown. This study characterized the in vitro contractile response of human and rat bladder SMC to several pharmacological agonists known to induce in vivo contraction of intact bladder muscle. MATERIALS AND METHODS: Human and rat bladder SMC were seeded separately within attached collagen lattices. Contractility of SMC was analyzed by measuring alterations in lattice diameter after exposure and release to the following contractile agonists: carbachol (10(-7)-10(-3) microM), calcium-ionophore (10 microM), lysophosphatidic acid (LPA) (1 microM), endothelin (0.1 microM), KCl (3.33 mmicroM) angiotensin II (10 microM), and serotonin (100 microM). Results were recorded as a mean reduction of the lattice diameter. In addition, immunohistochemical analysis for phenotypic markers of smooth muscle cell differentiation was performed on bladder SMC cultured within collagen lattices. Human palmar fascia fibroblasts, which have been previously well characterized by in vitro contractility and immunohistochemistry, were tested in parallel and used as controls for all the above experiments. RESULTS: Human SMC had significant contractile responses to calcium-ionophore (31% +/- 4 relative percent contraction, p <0.05), LPA (34% +/- 4, p <0.05), and endothelin (37 +/- 5%, p <05). There was no significant contraction in response to carbachol, angiotensin II, KCl, or serotonin. Rat bladder SMC had a similar contractile response but did not contract in response to endothelin. In contrast to human and rat bladder SMC, fibroblasts did not contract to calcium-ionophore. CONCLUSIONS: In vitro cultured bladder SMC demonstrate loss of contractile response to normal in vivo pharmacologic agonists. Both human and rat bladder SMC can be distinguished in vitro from fibroblasts based upon their lack of contractile response to calcium- ionophore. These results demonstrate the ability to further characterize cultured bladder SMC with in vitro contractility. Further characterization is essential if we are to advance our understanding of the clinical applicability of in vitro studies utilizing cultured bladder SMC.     

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.