【作者】 张永革；

【导师】 宋波；

【作者基本信息】 第三军医大学 ， 外科学， 2009， 博士

【摘要】 背景及目的探讨膀胱Cajal间质细胞(Interstitial cell of Cajal,ICC)在神经源性膀胱兴奋性改变中的作用。神经源性膀胱(Neurogenic bladder,NGB)是临床上常见的一种膀胱功能障碍,脊髓损伤是其常见原因之一。主要有两种类型:骶髓损伤(Sacral cord injury,SCI)和骶髓上损伤(Suprasacral cord injury,SSCI),骶髓损伤会导致逼尿肌无反射(Detrusor areflexia,DA),表现为排尿期膀胱不能收缩或者收缩无力;而骶髓上损伤会导致逼尿肌反射亢进(Detrusor hyperreflexia,DH),表现为储尿期出现膀胱自发或诱发的收缩。既往对神经源性膀胱发病机理的研究多进行的是神经病理学研究,传统理论认为,骶髓损伤的部位在骶髓初级排尿中枢,膀胱失去初级排尿中枢的控制,表现为排尿期不能出现收缩;骶髓上损伤的部位为骶髓以上,骶髓初级排尿中枢保存完整,失去高位中枢的控制,膀胱出现反射亢进的表现。但这些理论对神经病变后逼尿肌水平何种病变的下游机制解释不清,进一步的研究也发现,神经源性膀胱的膀胱壁也出现一系列的病理改变,包括肌细胞排列紊乱,分布不均匀;肌细胞间隙增宽或不均匀,弹性纤维减少,胶原纤维增生;中间连接减少,伴有缝隙连接的出现等等,但两种不同的病变都有类似的病理改变,显然还有我们未知的病理生理改变导致了膀胱功能的继发性改变。Cajal间质细胞是胃肠道平滑肌活动的起搏细胞,参与胃肠道基本电节律(Basic electrical rhythm,BER)的产生、传递电信号以及介导肠神经和平滑肌之间的信号调节。ICC细胞保持正常的结构、功能及分布,对胃肠动力的产生和功能调控至关重要,其分布密度的异常和细胞网络完整性破坏与一些胃肠动力紊乱性疾病如Hirschsprung’s病和慢性传输型便秘的发生密切相关。近年来国内外学者用相差显微镜、免疫组织化学分析和电镜技术研究人或动物具有自发性活动的平滑肌标本发现,ICC细胞普遍存在于各种器官,包括泌尿道的肾盂、输尿管和膀胱,但ICC细胞在泌尿道的作用还不清楚。研究发现泌尿道存在ICC细胞,且具有自发性兴奋的特征。进一步研究发现,在先天性肾盂输尿管连接部梗阻患者,肾盂输尿管连接部ICC细胞数量减少;在先天性巨结肠-巨膀胱-小肠蠕动失迟缓症患者,膀胱ICC细胞数量也是减少的;在人膀胱出口梗阻引起的过度活动膀胱中ICC细胞数量增加;本课题组的前期研究也发现,在大鼠膀胱出口梗阻引起的逼尿肌不稳定模型中,膀胱ICC细胞数量也明显增加,这些都提示ICC细胞可能在膀胱活动中起重要作用。因此,我们推测不同部位脊髓损伤后膀胱功能出现了典型的逼尿肌反射亢进和逼尿肌无反射表现,是否与ICC细胞的变化有关?ICC细胞特异性表达c-kit受体(Kit是一种编码酪氨酸激酶受体的原癌基因),Kit信号通路对细胞的发育和表型维持至关重要。甲磺酸伊马替尼(Glivec)是一个选择性的c-kit受体酪氨酸激酶抑制剂,经美国FDA批准用于治疗费城染色体阳性的慢性髓样白血病和c-kit阳性的胃肠道间质肿瘤。研究发现Glivec不仅可以抑制胃肠道ICC细胞的表达,还可以抑制离体豚鼠膀胱自发性活动和逼尿肌的自发性动作电位,说明ICC细胞可能参与了膀胱的自发性活动和逼尿肌的自发性动作电位。本课题拟通过对比研究骶髓上及骶髓损伤后逼尿肌反射亢进和逼尿肌无反射膀胱ICC细胞数量变化、兴奋性变化及用Glivec阻断从细胞水平、组织水平和在体水平阻断ICC后,观察ICC细胞兴奋性变化、肌条自发性活动的变化和膀胱功能的改变,探讨膀胱ICC细胞在逼尿肌反射亢进和逼尿肌无反射膀胱兴奋性改变中的作用。方法本课题以成年雌性SD大鼠为研究对象,建立骶髓及骶髓上损伤实验模型,围绕正常组、膀胱造瘘组、骶髓上损伤组及骶髓损伤组之间的对比进行以下研究:1、形态学研究:免疫荧光标记各组膀胱ICC细胞,观察各组膀胱ICC细胞数量的变化,探讨ICC细胞数量变化与膀胱兴奋性改变的可能关系。2、功能学研究:急性分离各组膀胱的ICC细胞,激光共聚焦显微镜检测膀胱ICC细胞自发性钙波变化、即细胞兴奋性的变化,观察ICC细胞兴奋性变化与膀胱兴奋性改变的可能关系。3、甲磺酸伊马替尼(Glivec)阻断ICC细胞对膀胱兴奋性的影响:①细胞水平:用Glivec阻断ICC细胞的c-kit受体,观察阻断c-kit受体后膀胱ICC细胞兴奋性的变化。②组织水平:进行不同组别的膀胱肌条实验,在实验过程中加入Glivec,观察肌条自发性收缩的变化。③在体水平:进行大鼠膀胱充盈性测压实验,经尾静脉注射Glivec,观察膀胱兴奋性改变。结果1、成功制作了脊髓损伤后逼尿肌反射亢进和逼尿肌无反射两种动物模型。2、形态学研究显示:骶髓上损伤大鼠膀胱ICC细胞数量较正常组和膀胱造瘘组明显增加,膀胱造瘘组和正常组大鼠膀胱ICC细胞无明显差异,骶髓损伤组膀胱ICC细胞数量较正常组和膀胱造瘘组减少。3、成功进行了大鼠膀胱ICC细胞的急性分离和鉴定。4、功能学研究显示:骶髓上损伤大鼠膀胱ICC细胞自发性兴奋性较正常组和膀胱造瘘组明显增加,膀胱造瘘组大鼠和正常组大鼠膀胱ICC细胞自发性兴奋性无明显差异,骶髓损伤组膀胱ICC细胞自发性兴奋性较正常组和膀胱造瘘组降低。5、Glivec可以以剂量依赖性的方式抑制膀胱ICC细胞的自发性钙波,低浓度(1×10-6mol/L)的Glivec对逼尿肌反射亢进大鼠膀胱ICC细胞自发性钙波有抑制作用,而5×10-5mol/L的Glivec对逼尿肌无反射大鼠膀胱ICC细胞自发性钙波有抑制作用。6、在一定的前负荷(1g)下,各组大鼠膀胱表现了不同的自发性收缩,逼尿肌反射亢进大鼠膀胱自发性收缩的幅度高于逼尿肌无反射大鼠膀胱。7、Glivec可呈剂量依赖性的方式降低膀胱逼尿肌肌条自发性收缩的幅度,低浓度(1×10-5mol/L)的Glivec对逼尿肌反射亢进大鼠膀胱逼尿肌肌条自发性收缩的幅度有抑制作用,而5×10-4mol/L的Glivec对逼尿肌无反射大鼠膀胱逼尿肌肌条自发性有抑制作用。任何浓度的Glivec对肌条自发性收缩的频率无影响。8、10mg/kg的Glivec可以增加逼尿肌反射亢进大鼠膀胱容量和顺应性,对逼尿肌无反射大鼠膀胱容量和顺应性无明显影响。20mg/kg的Glivec抑制逼尿肌反射亢进膀胱充盈过程中的自发性收缩。结论1、不同部位脊髓损伤引起的逼尿肌反射亢进和逼尿肌无反射大鼠模型中,膀胱ICC细胞数量和兴奋性出现了不同的变化。2、Glivec阻断ICC细胞后,抑制了逼尿肌反射亢进肌条的自发性收缩,增加了逼尿肌反射亢进膀胱容量和膀胱顺应性,并抑制了膀胱的不自主收缩;而不能抑制逼尿肌无反射肌条的自发性收缩,膀胱容量和顺应性无明显增加。3、膀胱ICC细胞数量增多和兴奋性的增加参与了逼尿肌反射亢进的功能改变,而ICC细胞数量的减少和兴奋性的降低可能与逼尿肌无反射有关。4、Glivec可增加逼尿肌反射亢进大鼠膀胱容量和顺应性,抑制膀胱不自主收缩,提示Glivec可能为逼尿肌反射亢进的治疗提供一个新的思路。更多还原

【Abstract】 Background and objectiveThe aim of this study is to investigate the role of interstitial cell of Cajal(ICC) in excitability change of neurogenic bladder. Neurogenic bladder, which is always caused by spinal cord injury, is a common bladder dysfunction. According to the segment of lesion, spinal cord injury can be classified as sacral cord injury(SCI) and Suprasacral cord injury(SSCI). SCI induce detrusor areflexia(DA), which displays contraction dysfunction or atony in bladder during voiding phase. SSCI induce detrusor hyperreflexia (DH), which displays spontaneous or evoked contraction in bladder during filling phase. According to the traditional theory, the bladder without the control of primary micturition center may display no contraction during voiding phase when the injury locates at sacral cord primary micturition center. The bladder under the control of primary micturition center and without the control of high centeral may display hyperreflexia when the injury locates at upper sacral cord. However, these theories can not explain the further changes of detrusor after nerve injury, such as muscle cells arranging disorder and maldistribution, intercellular space widening or nonuniform with decrease of elastic fibers and increase of collagen fibers, or decrease of intermediate junction following with appearance of gap junction. Moreover, SCI and SSCI always have the similar pathological change, which indicates we have not get enough data on the pathophysiological changes inducing the changes of bladder function.ICCs act as the pacemaker of smooth muscle activation in gastrointestinal tract. They play a role in producing gastrointestinal basic electrical rhythm(BER), transmitting electrical signal and mediating the signal between intestinal nerve and smooth muscle. It’s important for ICCs to maintain normal structure, distribution and function to regulate gastrointestinal motility. The abnormal distribution of ICCs and the destroy of cellular network may related with gastrointestinal motility disorders, such as Hirschsprung’s disease and chronic transmitting constipation. Recently, several investigators found that there are ICCs in many organs, such as renal pelvis, ureter and bladder, using phase contrast microscope, immunohistochemical anlysis and electron microscopy. However, the role of ICCs in urinary tract is still unknown. Studies demonstrated that ICCs in urinary tract presented spontaneous excitability. Some further studies indicated that the number of ICCs in the pyeloureteral junction decreased in patients with congenital pyeloureteral junction obstruction, and the number of ICCs in bladder decreased in patients with megacystis-microcolon intestinal hypoperistalsis syndrome. The munber of ICCs increased in overactivity bladder with the bladder outlet obstruction. Previously we found that ICCs increased significantly in bladder with BOO in rats model, which indicated ICCs may contribute to bladder activity. Therefor, we speculate that ICCs may contribute to detrusor hyperreflexia or detrusor areflexia in neurogenic bladder.C-kit receptors, which determined by a proto-oncogene kit encoding tyrosine kinase receptors, specially express in ICCs. The signal mediated by kit is important to maintain the development and function of cells. Imatinib mesylate (Glivec) is a selective inhibitor of c-kit receptor tyrosine kinases, and has USA Food and Drug Administration approval for the treatment of Philadelphia chromosome-positive chronic myeloid leukaemia and c-kit positive gastrointestinal stromal tumours. The data demonstrate that Glivec block kit signaling expression in gastrointestinal tract, and can suppress the spontaneous activity in bladder and the spontaneous action potential in detrusor in guinea pigs, suggests that ICCs contribute to the spontaneous activity in bladder and the spontaneous action potential in detrusor.The present study purposes to investigate the role of ICCs in the changes of detrusor hyperreflexia and detrusor areflexia in the bladder. We detect the changes of quantity and excitability of the ICCs in rats with detrusor hyperreflexia and detrusor areflexia, respectively. And we observe the changes of excitability of ICCs, the spontaneous activity of strips and bladder function, using c-kit receptor inhibitor Glivec to block ICCs in vitro and in vivo.MethodsFemale SD rats were used in the present study. These rats were randomly divided into normal group, post-cystostomy group, SSCI group and SCI group. The following studies were performed: 1. The bladders of rats were harvested in 4-6 weeks after spinal cord injury or cystostomy. The number of ICCs in each group was counted by immunofluorescence staining. 2. The ICCs of bladder in each group were freshly dispersed and the spontaneous calcium waves of ICCs were detected using laser confocal microscopy. 3. The changes of the spontaneous calcium waves of ICCs, the spontaneous contraction of the bladder strips and the changes of bladder function were detected after Glivec blocked the ICCs in each group.Results1、We successfully create the rat model with detrusor hyperreflexia and detrusor arereflexia.2、There are significantly more ICCs in bladder of SSCI rats than that in normal and post-cystostomy rats bladder. The quantity of ICCs in normal bladder is similar to that in post-cystostomy bladder. There are significantly less ICCs in bladder of SCI rats than that in normal and post-cystostomy rats bladder.3、We successfully freshly disperse and identify ICCs in rats bladder.4、The spongtanous excitability of ICCs in SSCI rats is higher than that in normal and post-cystostomy rats. The spongtanous excitability of ICCs in normal rats is similar to that in post-cystostomy rats. The spongtanous excitability of ICCs in SCI rats is lower than that in normal and post-cystostomy rats.5、The spontaneous calcium waves of ICCs in bladder is suppressed by Glivec with dose dependent. Glivec in low concentration(1×10-6mol/L) suppress the spontaneous calcium waves of ICCs in bladder with detrusor hyperreflexia, while 5×10-5mol/L Glivec suppress the spontaneous calcium waves of ICCs in bladder with detrusor areflexia.6、Each group has different spontaneous contractions of bladder in rats with unvarying preload(1g). The amplitude of spontaneous contractions of bladder with detrusor hyperreflexia in rats is higher than that in bladder with detrusor areflexia.7、The amplitude of spontaneous contraction of detrusor strips is inhibited by Glivec application with dose dependent. Glivec in low concentration(1×10-5mol/L) suppress the amplitude of spontaneous contraction of detrusor strips with detrusor hyperreflexia in rats, while 5×10-4mol/L Glivec suppress the amplitude of spontaneous contraction of detrusor strips with detrusor areflexia.8、Glivec(10mg/kg) increase the bladder capacity and compliance in rats with detrusor hyperreflexia and can not influence the bladder capacity and compliance in rats with detrusor areflexia. The spontaneous contraction of bladder can be suppressed by Glivec(20mg/kg) during the storage phase.Conclusion1. ICCs in rat bladder with spinal cord injury, including SSCI and SCI,occur to the changes of quantity and excitability.2. Glivec can suppress the spontaneous contraction of detrusor strips, increase the bladder capacity and compliance, and inhibit the spontaneous contraction of bladder with detrusor hyperreflexia by blocking ICCs, whereas it can not suppress the spontaneous contraction of detrusor strips, increase the capacity and compliance of bladder with detrusor arereflexia by blocking ICC.3. The increase of ICCs quantity and excitability may contribute to the bladder function of detrusor hyperreflexia, and the decrease of ICCs quantity and excitability may involve in the bladder function of detrusor arereflexia.4. Glivec increase the bladder capacity and compliance, and inhibit the spontaneous contraction of bladder with detrusor hyperreflexia. These results indicate that Glivec may be a new strategy for the clinical treatment of detrusor hyperreflexia.更多还原