【作者】 何鹏；

【导师】 宋波；

【作者基本信息】 第三军医大学 ， 外科学， 2012， 博士

【摘要】 背景与研究目的：膀胱兴奋性的改变在多种排尿功能障碍性疾病的发生发展中起着不可忽视的作用。而导致膀胱兴奋性变化的原因很多，如梗阻、炎症、神经病变等，其中以膀胱部分出口梗阻(partial bladder outlet obstruction，PBOO)和其他中枢及外周神经系统病变多见，是导致尿频、尿急、甚至急迫性尿失禁等膀胱过度活动(overactive bladder，OAB)症候群的重要原因。目前对OAB的发病机制尚未完全阐明，临床针对其治疗主要还是以胆碱能神经受体作为主要靶点，辅助应用肾上腺素能受体阻断剂及钙离子通道阻断剂配合治疗。面对部分由非神经因素造成的特发性OAB患者，该治疗效果欠佳。毫无疑问，对膀胱兴奋性的研究成为当前领域的研究热点。膀胱主要受神经系统的支配，同时，也具有不依赖神经因素的自发性兴奋性。对各种病理状态下的膀胱兴奋性改变的认识，目前学术界存在着两派学说，即以WC deGroat为代表“神经源性学说”和以AF Brading为代表的“肌源性学说”。前者长期以来强调，膀胱的活动是由中枢及外周神经系统控制的，其发生的一系列兴奋性及收缩性的改变均与神经有着密切联系，主要表现在传入传出神经或神经元的变化、各种神经递质的产生和分泌变化以及各种神经受体数量和功能的变化。但后者认为，除开神经的影响因素，逼尿肌本身兴奋性的变化、逼尿肌细胞之间通讯的改变、各种化学和牵张感受细胞的生理功能变化也参与了膀胱兴奋性的调控。目前，“肌源性”学说作为传统“神经源性”学说的重要补充已逐步被学术界认可，在膀胱兴奋性调控领域的影响日渐增加。膀胱卡哈尔间质细胞(interstitial cell of Cajal，ICC)的发现，使膀胱来源的因素在膀胱兴奋性调控中的作用逐渐受到重视。膀胱ICCs是2002年首先被英国学者McCloskey在豚鼠中发现的一类与胃肠道起搏细胞ICC类似的特殊间质细胞。膀胱ICCs的具体生理作用目前还有争议，从最初的参与膀胱活动的起搏，到神经信号的整合及传递，以及作为感受器将各种刺激上传，都进行了深入的研究。而离子通道，这种参与细胞兴奋性调控最直接的组件，在膀胱中，尤其是膀胱平滑肌(bladder smoothmuscle cells, BSMCs)和ICCs上，被作为中心环节进行了诸多研究，也是目前膀胱兴奋性的研究热点。膀胱ICCs维持其功能及发挥作用依赖于细胞膜及胞内分布的各种离子通道。目前在膀胱ICCs上已经发现的通道非常多，与细胞兴奋性和收缩性直接相关的包括电压门控钙通道、大电导钙激活钾通道、ATP依赖的钾通道、快速激活的钠通道、氯离子通道等。而在自发节律产生中占有绝对关键地位的超极化激活环核苷酸门控阳离子(hyperpolarization-activated cyclic nucleotide-gated, HCN)通道在可兴奋细胞中的研究才刚刚起步，只是在神经元及窦房结中得到了深入探讨，而其他组织中的各种重要可兴奋细胞上的研究还只停留在初级阶段。HCN通道对于细胞兴奋性的维持和调节可能具有重要且深远的意义。HCN通道是最初在心脏和神经元中发现的一类具有特殊生理特性的离子通道，他和其他众多在去极化阶段激活的通道相反，在超极化阶段才能激活，并引发阳离子内流，HCN通道具有普通离子通道不具备的一些特殊的特性，比如复杂的双门控调节、混合离子通透性、微弱的单通道电导等，而其产生的Ih电流呈电压依赖性，除了被普遍认为是起搏活动必须的“起搏电流”外，还具有稳定细胞膜电位、参与可兴奋细胞自律性的调节、调节神经递质释放、参与各种感觉、视觉、听觉信号整合等基础生理功能。HCN通道是否在胃肠道ICCs中参与其慢波电位的形成也是学者们最为关注的方面，在窦房结以外的细胞和组织中，HCN通道产生的Ih电流能否成为pacemaker的主要构成成分都是亟待解决的问题。膀胱兴奋性改变在膀胱病理状态下导致的一系列临床症状有直接的联系，膀胱ICCs作为新近发现的一类特殊间质细胞，在泌尿系统中具有多种生理功能，且与膀胱兴奋性调控密切相关。而同样参与多种生理功能调控的HCN通道及其亚型是否参与膀胱兴奋性调控是一个非常有趣而且大胆的设想，目前世界上还没有针对这个方面进行研究的团队。首次将膀胱ICCs上的HCN通道作为膀胱兴奋性研究的切入点具有一定的前瞻性，可为膀胱兴奋性调控研究提供新的思路，并为临床治疗排尿功能障碍性疾病提供了潜在的作用靶点，丰富泌尿系统病理生理的理论基础。基于膀胱兴奋性的调节，本研究通过分子生物学和形态学方法对HCN通道及其亚型在膀胱ICCs中的表达及分布进行观察，通过功能学手段对HCN通道参与ICCs兴奋性调控和膀胱兴奋收缩的机制进行了初步探索，并对膀胱ICCs上的HCN通道的基本生理特性进行了初步研究。材料及方法：本研究采用2-3月龄大鼠作为主要研究对象，首先利用RT-PCR和Western blotting技术对正常膀胱组织中HCN通道1-4四个亚型的表达进行研究；然后通过免疫荧光技术阐述该通道四个亚型在膀胱中的具体分布情况；通过金属离子荧光探针负载技术观察培养的ICCs胞内钙离子和钾离子浓度在HCN通道阻断剂作用下的变换，以及对该通道阻断剂对离体膀胱逼尿肌肌条牵张诱发的节律性收缩的影响，从而了解该通道在膀胱兴奋收缩中的作用；最后利用膜片钳技术对急性分离的ICCs上的HCN通道电流进行检测，并记录特异性阻断剂下该电流的变化情况。结果：1. RT-PCR发现膀胱组织中HCN1-4四个亚型的mRNA均有表达，与心脏和小肠类似；WB检测发现膀胱组织中HCN1-4四个亚型的蛋白均有表达，利用灰度值进行半定量分析发现HCN1亚型表达比例最高。2.激光共聚焦显微镜观察正常大鼠膀胱中HCN阳性和c-kit阳性的细胞有共存现象，与myosin阳性的细胞无共存现象。3.膀胱ICCs中表达的HCN亚型以HCN1、HCN2、HCN4为主，并未检测到HCN3亚型表达。4. HCN通道阻断剂ZD7288能使静息状态下的膀胱ICCs胞内钙一过性不明显上升，但能显著降低高钾诱发的胞内钙离子浓度升高。5. ZD7288能显著降低静息状态下的膀胱ICCs胞内钾离子浓度，但拮抗高钾诱发的胞内钾离子浓度上升效果不明显。6. ZD7288可显著降低正常逼尿肌肌条的自发性收缩幅度，但小幅度上调了收缩频率，且ICCs特异性拮抗剂Glivec作用后收缩现象无明显变化。7.成功获得急性酶分离的大鼠膀胱ICCs，并在该细胞上记录到典型的Ih电流轨迹，ZD7288作用10min后效果稳定，能显著降低该电流强度。结论：1.正常大鼠膀胱中HCN通道主要表达在膀胱ICCs上，亚型以HCN1、HCN2、HCN4为主，未发现HCN3亚型表达。而平滑肌上则没有HCN通道表达。2. HCN通道与膀胱ICCs的兴奋密切相关，阻断该通道能降低兴奋时胞内的钙离子浓度。且该通道参与了稳定膀胱ICCs静息状态下膜电位的作用，ICCs兴奋时该通道阻断剂使胞内钾离子浓度下降不明显说明细胞K+内流并不是主要依靠HCN通道完成。3. HCN通道可能通过膀胱ICCs影响膀胱肌肉的兴奋性及收缩性，而非膀胱逼尿肌，且主要通过Ih电流参与各种功能的调控。更多还原

【Abstract】 Background and Objective：Change of bladder excitation was the most important reason for the generation anddevelopment of micturational dysfunctions. Obstruction, imflammations and nervousdiseases were candidate for the alteration of excitability of urinary bladder. Overactivebladder(OAB) secondary to the partial bladder outlet obstruction (PBOO) and otherdiseases of central and peripheral nervous system is one of the most common micturationaldysfunction in urology which was represented by urinary frequency, urinary urgency, andeven stress urinary incontinence. The exact pathophysiologic mechanism is unknown untilnow. Cholinergic receptors were chose as pharmacol target in treatment in the clinic aspectstoward OAB. Anti-adrenonergic receptor agents and calcium channel blocker are used alsoas assistance and show effective results. However, it’s hard to solve the idiopathic OABcaused by non-neurogenic factors and the treatment for it is not effective enough. There isno doubt that researches on bladder excitability will become a hot spot in this field.Urinary bladder is innervatied by nervous system, while self-excitation without thecontrol of nerve is also a special function of vesicles. Two theories were wide-spreaded inthe world on the changes of bladder excitation in several pathologic statuses.“Neurogenictheory” proposed by W.C. de Groat emphasized that all activities of bladder were controlledby central and peripheral nervous system. Series of excitatory and contractile changes wereclosely associatied with nerves. Disturbance of afferent and efferent nerve fibers,neurotransmitter secretion problems and neuroreceptor alterations were candidate for it.Nevertheless,“myogenic theory” proposed by A.F. Brading indicated that changes ofdetrusor excitation, cell-to-cell communication and the physiological functions of chemicaland mechanical stretch reception cells participate in the regulation of bladder excitation. “Myogenic” theories were widely accepted by scientists all over the world as a bestcomplementary for “neurogenic” theories in the field of bladder excitation regulations andget more and more concerns.“Myogenic/cystogenic” factors in the controlling of bladder excitation gain much moreinterests right after the charactization of interstitial cells of Cajal in bladder which was firstfound in guinea-pig bladder by a British scientists McCloskey. However, the physiologicalfunctions of bladder ICCs are still controversial and gain many attentions of researchers.From the initiation of bladder activity, integration and transmission of neurosigmals toupload the informations as stimulation receptors, there are still a long way to go. Ionchannels, which were considered as direct controlling module in bladder, especially onbladder smooth muscle cells and interstitial cells of Cajal, were investigated by severalresearch groups as a vital component.Maintain and operation of functions in bladder ICCs rely on the various ion channelslocated on the cellular membrane and intracellular aspects. Lots of ion channels have beenidentified in bladder ICCs, including voltage-gated calcium channels, BK channel,ATP-sensitive potassium channel, rapid sodium channel and chloride channels which wereassociated with cellular excitation. Hyperpolarization-activated cyclic nucleoted-gated(HCN) channel, first found in sinoatrial node and neurons, become more and more“popular” to the scientists because it was associated closely with autorhythmicity. Studiesof HCN channels on excitatory cells were just at the beginning stage and most of theapproaches were applied in neurons and sinoatrial node. Experiments on other excitatorycells expected more investigations and may explore some important significance.HCN channel was a special ion channel firstly found in heart and neurons. Unlike theactivation mode of normal channels which opened in the depolarization period, HCNchannels opened in the hyperpolarization stage and generated an inward cation current. Theunique properties such as complicatied dual-gate mechanism, permeation of admixture ionsand weak single channel conductance were different from most of the other channels. Thecurrent Ih generated by HCN channels was considered to be a pacemaker current inpacemaking activities. In addition, Ih also plays an important role in several basicphysiological functions such as stabilizing the resting membrane potential, regulating theautorhythmicity and controlling the release of neurotransmitters in excitatory cells. Wether Ih current form the slow wave in the gastrointestinal tract is a good and complicatiedquestion.Generally speaking, excitation changes of bladder have a close connection with seriesof clinical symptons secondary to the bladder diseases. The newly found bladder ICC was aspecial interstitial cell and functions a lot in urinary tract and was considered to be a keyfactor in modulation of bladder excitation. It is interesting that we hypothesis themulti-functional HCN channels was located on multi-functional ICCs in bladder, and selectHCN channel and its subtypes as a breakpoint to understand the bladder excitation. So far,few studies have focused on the HCN channels located in the urinary bladder, especiallytheir biophysical properties and their role in cell excitation. Researches about HCNchannels on bladder ICCs may reveal a new pathophysiological theory in urology andprovide new ideas on excitation investigations.Materials and Methods：Female Sprague–Dawley rats (weighing180-220g) were used in studies. RT-PCR andWestern blotting were taken to investigate the transcripts and protein expression of HCN1-4.Double labeled immunofluorescent study was performed then to identify the location ofHCN channel and its subtypes in bladder. Change of cytoplasmic Ca2+and K+combinedwith fluorescent molecular probes was observed to understand the effects of HCN channelblocker on ICCs. Bladder smooth muscle was studied as isolated strips and specific channelinhibitor ZD7288was then applied to characterized the effect of HCN channel and itsmechanism in regulating bladder contraction. Last but no the least, patch clamp techniquewas used in enzymed-dispersed bladder ICCs and Ih current can be recorded in thewhole-cell mode. HCN channel blocker was also applied to explore the changes of thecurrent.Results and Discussion：1. We detected mRNA corresponding to all four of the HCN channel subtypes in theurinary bladder of the rat. The heart and intestine were used as positive controls. All HCNchannel isolations protein can be detected in bladder tissue using Western blotting. Relativeexpression level of the HCN channel subtypes showed the expression of HCN1issignificantly higher than that of other subtypes.2. Within the individual cell bodies, the HCN isoform and kit immunostaining were uniformly present throughout the cytoplasm, but no co-labeling of HCN1-4and smoothmuscle was observed.3. There was no apparent staining for the HCN3subtype in the interstitial regions.HCN1, HCN2and HCN4were the prominent isolations locatied on bladder ICCs.4. Specific HCN channel blocker ZD7288raise the concentration of intracellular Ca2+in an inapparent level of bladder ICCs at resting state, but reduced the concentration ofintracellular Ca2+induced by a higher concentration of extracellular K+significantly.5. Concentration of cytoplasmic potassium can be depressed by ZD7288at restingstate. But ZD7288reduce the concentration of K+induced by a higher extracellular K+wasinapparent.6. The amplitude of the detrusor strips can be reduced by selective HCN channelblocker ZD7288, while contractile frequency was raised by it. Application of Glivec, whichwas a significant ICC inhibitor, makes no difference in contraction followed up withZD7288.7. Bladder ICCs can be isolated by enzyme and used in electrophysiological studies.Typical Ih current traces were recorded and ZD7288can significantly reduce the currentamplitude.Conclusion：1. HCN channel and its subtypes were located on bladder ICCs, neither on BSMCs.HCN3subtype was not expressed in native rat bladder. Other isolations can be found in allregions of urinary bladder.2. HCN channel was closely associated with cellular excitation of bladder ICCs.Intracellular Ca2+can be reduced by using HCN channel blocker when ICCs were activatedand HCN channel participate in stabilizing resting membrane potentials. Influx ofpotassium was not carried out by HCN channel.3. HCN channel can affect the excitation and contraction of bladder, presumably viabladder ICCs and the regulation of series cellular functions probably was carried outthrough Ih current.更多还原