【作者】 贾卓敏；

【导师】 宋波；

【作者基本信息】 第三军医大学 ， 外科学， 2009， 博士

【摘要】 背景及目的:膀胱是具有高度顺应性的空腔器官,其排尿功能是通过膀胱收缩、尿道括约肌松弛协调完成的。膀胱收缩功能异常疾病临床发病率高,严重影响患者生活质量,但治疗效果不理想。究其原因是膀胱收缩功能调节复杂、相关疾病的发病机理不清。膀胱功能主要依赖于中枢神经系统（ central nervous system;CNS ）的控制,通过胆碱能神经调节收缩,肾上腺素能神经调节舒张,非胆碱能非肾上腺素能神经起辅助作用。但是在膀胱收缩异常的疾病的临床治疗中应用神经调节药物,症状虽有部分缓解,但是不能彻底治愈。提示神经调节为基础的理论不能满意解释膀胱收缩功能异常疾病的产生机制。离体膀胱可以产生局部的微小收缩或整体收缩,而整体收缩也可表现出膀胱内压力的变化,另外离体逼尿肌条也观察到其存在自发收缩活性。说明膀胱在神经调控外,其本身具有的特征也参与了膀胱收缩功能的调节,据此我们中心根据多年研究提出“膀胱源性”学说。膀胱ICC细胞是近年来在人和豚鼠等动物泌尿系统的输尿管、膀胱以及前列腺等组织内发现的形态上与胃肠道Cajal间质细胞（interstitial cells of Cajal,ICC）类似的细胞。胃肠道ICC是一种特殊的间质细胞,对胃肠道基本电节律的形成、神经传导的调控、信号整合等多种生理功能起到了重要的作用。胃肠道ICC保持正常的结构、功能及分布,对胃肠动力的产生和收缩功能调控至关重要,其分布密度减少和细胞网络完整性被破坏与一些胃肠动力紊乱性疾病的发生密切相关。而膀胱ICC细胞的研究尚处起步阶段,McCloskey等提出膀胱ICC细胞对膀胱的收缩功能调节可能具有重要作用。但是膀胱收缩是由全部逼尿肌细胞协调活动完成的,而膀胱ICC细胞不能在完全独立状态下实现对膀胱收缩功能的调节,那么膀胱ICC细胞必然同神经存在某种联系?胆碱能神经在膀胱的神经调控中起主要作用,其作用的实现是通过释放乙酰胆碱,作用于膀胱毒蕈碱样胆碱能受体（muscarinic receptor,M受体）,引起逼尿肌收缩。逼尿肌M受体变化在各种膀胱收缩功能异常疾病的发病机理中具有重要作用。Braverman等在神经源性膀胱的研究中发现,M受体总量的增加或M2受体密度的增加,改变了大鼠去盆神经支配的膀胱逼尿肌的收缩特性,提示M₂受体变化可能主要致病因素。逼尿肌不稳定是最常见的排尿功能障碍性疾病,其发病机理可能与M受体亚型上调及M₂受体、M₃受体亚型之间的比例失调有关。最近的研究发现胃肠道ICC可以表达一系列神经受体,如嘌呤能受体、缓激肽受体、胆碱能和前列腺素受体。Kim等研究发现胆碱能刺激可以增加胃窦部和体部ICC的起搏频率,并且其胆碱能效应是由M₃受体介导的,提示胆碱能递质可以调节胃肠道ICC的兴奋性并通过ICC调节胃肠道平滑肌的收缩功能。本课题组前期研究发现,膀胱ICC细胞膜上有嘌呤能受体的表达,嘌呤能神经递质ATP可以兴奋膀胱ICC细胞。那么,M受体的激活是否可以兴奋膀胱ICC细胞,膀胱ICC细胞是否参与了胆碱能神经对膀胱收缩功能的调控?目前国内外未见相关报道。本课题拟围绕M受体亚型在膀胱ICC细胞中的分布,M受体的激活对膀胱ICC细胞兴奋性的影响以及阻断膀胱ICC细胞对膀胱收缩功能的影响三个方面进行研究。从一个全新的层面探讨膀胱收缩功能的调控机制,具有十分重要的理论价值,为膀胱收缩功能异常的研究提供新的切入点。方法:本课题以成年SD大鼠为研究对象。⑴形态学观察:通过免疫荧光双标和激光共聚焦显微镜观察的方法,鉴定大鼠膀胱ICC细胞的分布及膀胱ICC细胞上毒蕈碱样胆碱能受体五个亚型的表达;⑵细胞水平检测M受体对膀胱ICC细胞的功能的影响:进行大鼠膀胱ICC细胞和逼尿肌细胞原代混合培养,应用Fluo-3 AM钙荧光指示剂、胆碱能受体激动剂、亚型拮抗剂和激光聚焦显微镜观察膀胱ICC细胞[Ca²⁺]_i的变化,了解M受体对膀胱ICC细胞兴奋性的调节;⑶组织水平:从卡巴可对正常大鼠离体膀胱肌条收缩功能的影响、M₂和M₃受体亚型特异性阻断剂对膀胱肌条收缩功能的影响、应ICC细胞特异性阻断剂甲磺酸伊马替尼（Glivec）阻断膀胱ICC细胞后,卡巴可对正常膀胱肌条收缩功能影响的改变等不同角度,来探讨膀胱ICC细胞在膀胱收缩功能调节中的作用。结果:1.激光共聚焦显微镜下观察显示,SD大鼠膀胱内可见c-kit染色阳性的膀胱ICC细胞。2. SD大鼠膀胱ICC细胞仅表达M₂和M₃受体亚型。3.成功进行了大鼠膀胱ICC细胞和DSMC的原代混合培养及鉴定。4. M受体激动剂卡巴可作用膀胱ICC细胞后,可以引起[Ca²⁺]_i显著升高。5. M₂受体拮抗剂美索曲明可以轻度抑制卡巴可对膀胱ICC细胞的兴奋作用;M₃受体拮抗剂4-DAMP可以显著抑制卡巴可对膀胱ICC细胞的兴奋作用。6.在一定前负荷下（0.75g）,膀胱肌条出现自发性的期相性收缩,不同终浓度的卡巴可可以引起肌条出现整体收缩幅度及自发性收缩振幅显著增强,自发性收缩频率出现先升高后降低的趋势。7.美索曲明可以轻度抑制卡巴可引起的膀胱肌条自发性收缩幅度和频率和肌条收缩张力;4-DAMP可以显著抑制卡巴可引起的膀胱肌条自发性收缩幅度、频率和肌条收缩张力。8. Glivec可以呈浓度依赖方式抑制卡巴可对膀胱肌条的收缩作用,并且对卡巴可引起的膀胱肌条收缩效应产生舒张效应。9.应用Glivec阻断膀胱ICC细胞后,卡巴可对膀胱肌条的自发性收缩振幅和收缩效应均被减弱。结论:1.大鼠膀胱ICC细胞细胞膜上存在M₂受体和M₃受体。2.胆碱能递质主要通过M₃受体兴奋膀胱ICC细胞。3.膀胱ICC细胞可能参与了胆碱能神经对逼尿肌收缩功能的调控。更多还原

【Abstract】 Background and objective:Bladder, an empty organ with high compliance, always requires a coordinated contraction of the detrusor and relaxation of the urethra. Bladder dysfunctions are very common in clinic. Patients suffer from unbearable symptoms and there are no effective therapies to them in some patients because the regulation of bladder contractile function is complex and the pathogenesis of these diseases is not clear.Physiological contraction and relaxation of the urinary bladder are predominantly controled by central nervous system（CNS）. The bladder contraction is mainly regulated by cholinergic nerve and the relaxation is mainly regulated by adrenergic nerve combining with the assistance of non-adrenergiccholinergic nerve. Unfortunately, satisfactory therapeutic effect is seldom obtained when treating the diseases of bladder constriction disorders using several kinds of antagonists to nerve regulation, which suggests that the theory based on neuroregulation can’t explain the exact mechanisms underling bladder contractile function disoders. Previous studies have demonstrated that the isolated bladder had localized micromotion or generalized contraction,and the generalized contraction induced the change of intravesical pressure. It was also found that detrusor muscle strip could generate spontaneous contraction. Then we supposed a hypothesis based on intrinsic bladder changed to explain the disorders besides nerve regulation, and tried to find the pathogenesis of them for many years in our reseach center.Recently, some specialized cells with various morphological, electrophisiological and immunohistochemical characteristics of interstitial cells of Cajal （ICC） have been located in many regions of ureter, bladder, prostate and so on. ICC, the pacemaker cells of the gastrointestinal tract, play important roles in the formation of basic electric rhythm of gastrointestinal tract, the regulation of neural conduction, signal integration and so on. The normal structures, function and distribution are important to maintain the gastrointestinal motility and contractile function. The number of ICC decreaseing or damage of the cellular network may induce some gastrointestinal motility disorders. McCloskey et al found ICC may play important roles in the regulation of bladder contraction. Moreover, the location of bladder ICC have connections with intramural nerves and associated with smooth muscle cells. Then, there may be some special relationships between ICC and nerve in bladder.Cholinergic nerve play the predominant role in nerve regulation in bladder via released acetylcholine combining muscarinic receptor to induce detrusor contraction. Muscarinic receptors are related with many abnormalities of bladder contraction. Braverman et al found that total amount of M receptor or density of M₂ receptor increasing were associated with the contractility ability of bladder and those changes were independent with pelvic nerve, which suggests that M₂ receptor may be the main causative factor to this disease. Instability of detrusor muscle is the most common urination dysfunction; its pathogenesis may be related with the up-regulation of subtype of M receptor and disproportion of M₂ and M₃ receptor subtypes.Recent studies demonstrated that ICC in gastroinstestinal tract expresses several neuroceptors, such as purinergic receptors, bradykinin receptors, cholinergic and prostaglandin receptors. Kim et al found that the cholinergic stimulus mediated by M3 receptor increased the pacing frequency of ICC in gastic antrum and body, which suggests that cholinergic transmitter regulates the excitory of ICC in gastrointestinal tract to control the contractile function of smooth muscle in gastrointestinal tract. Our previous studies found that the ICC in bladder express purinergic receptors and ICC in bladder can be activated under the stimulation of purinergic neurotransmitter ATP. Then we are interested in the correlation between activation of M receptor and excitability of ICC in bladder, and whether ICC play roles in the regulation of bladder contraction underlying cholinergic nerve? There are no data on this subject reported by researchs of internal and abroad.This study will investigate the distribution of M receptor subtypes in bladder ICC, the relations of activation of M receptor and the excitory of ICC, and the contractile function of bladder when ICC were inhibited by Glivec, a specialized inhibitor of c-kit tyrosine kinase. The mechanism of regulation underling bladder contraction will be investigated by a firenew point, which is a worthy theory to offer new therapies for abnormalities of bladder contraction. Materials and MethodsIn this study, the roles of M receptors in bladder contractile function were observed in adult SD rats.⑴We identified the area of ICCs and the expression of the five muscarinic Cholinergic Receptors （M₁-M₅） on ICCs in bladder of rats using couple immunofluorescences and observing with confocal laser microscopy.⑵We investigated the contribution of M receptors to the function of ICCs in mixed primary culture of ICCs and detrusor muscle cells. Then the changes of [Ca²⁺]_i in ICCs were observed under confocal laser microscopy after using Fluo-3 AM Calcium fluorescence indicator, Cholinergic receptor agonist and subtypes antagonist to examine how M receptors regulate the excitability of ICCs.⑶The role of ICCs in bladder contractile function was researched by comparing the changes of contractile functions using Carbachol in normal rat bladder in vitro, M₂ and M₃ receptors hypospecificity blocking agents in bladder strips. Furthermore the bladder strips contractile properties induced by Carbachol application were detected after been exposed to Glivec to block c-kit and the normal function of ICCs.Results1. C-kit positive ICCs were found in bladder of SD rats under confocal laser microscopy.2. We found that M₂ and M₃ but not M1, M4 and M5 receptor subtypes were expressed in bladder ICCs of SD rat by double lable of immunofluorescence.3. We successfully identified bladder ICC in mixed primary culture.4. In vitro application of Carbachol, an agonist of M receptor, significantly enhanced [Ca²⁺]_i in cultured ICC.5. The Carbachol-induced excitability in cultured ICCs was mildly inhibited by Methoctramine, the antagonist of M₂ receptor, while it was significantly inhibited by 4-DAMP, the antagonist of M₃ receptor.6. The bladder strips showed spontaneous contractions under a fixed tention（0.75g）. Gradually increasing the concentration of Carbachol could induce significant increasing in the amplitude of whole contractions and spontaneous contractions. Moreover, the frequency of spontaneous contractions showed high frequency in first and then down later.7. The amplitude/frequency of spontaneous contraction and the tention of the strips induced by Carbachol could be significantly inhibited by 4-DAMP but mildly inhibited by Methoctramine.8. The contraction of bladder strips could be inhibited by concentration dependent Glivec. Glivec could relax the bladder strips which were in contraction state induced by Carbachol.9. Glivec could significantly decrease the amplitude of spontaneous contraction and contractile effect of bladder strips induced by Carbachol.Conclutions1. ICCs express M₂ and M₃ receptors in bladder of rats.2. Cholinergic nerve stimulate can excite ICCs in bladder mainly via M₃ muscarinic receptors.3. ICCs in bladder might play roles in the regulation of detrusor contraction by the interaction with cholinergic nerve.更多还原