【作者】 卫中庆；

【导师】 严春寅；

【作者基本信息】 苏州大学 ， 泌尿外科学， 2009， 博士

【摘要】 糖尿病是一种常见的内分泌代谢疾病,糖尿病性神经病变是糖尿病慢性并发症中发病率最高的一种,糖尿病膀胱病( diabetic cystopathy, DCP)是糖尿病外周神经病变的一种,属于神经源性膀胱尿道功能障碍(Neuropathic dysfunction of bladder,NB)。作为糖尿病引起的泌尿系统并发症,其发病率占糖尿病患者的50%左右。临床研究显示即使对于那些血糖控制稳定的患者,仍有25%的DCP发病率。迄今DCP的确切发病机制尚不完全清楚,现研究认为主要是:①糖尿病引起的外周和自主神经病变,②同时还存在着肌源性异常等其它因素。寻找有效的方法,尽早积极预防和治疗糖尿病膀胱病变,改善下尿路症状,是糖尿病患者综合治疗及提高生活质量的重要组成部分。目前糖尿病神经病变的治疗主要集中在药物治疗,药物治疗虽有一定效果,但副作用不可忽视。另外,有创治疗包括间歇清洁导尿、膀胱造瘘术、尿道膀胱颈切开等方法,可缓解大量剩余尿的问题,改善剩余尿造成的尿路感染和上尿路损伤,但治标不治本,且处理不当易招致进一步的感染。如何找到有效、安全的预防、治疗DCP病变,解除DCP逼尿肌无力、剩余尿多,一直是人们在探索的问题。早期积极预防DCP的发生和发展,是糖尿病患者综合治疗及提高生活质量的关键所在。使用体外电脉冲刺激(Transcutaneous Electrical Nerve Stimulation ,TENS)技术治疗DCP报道甚少,且尚无其尿动力学及机制方面的研究。另研究发现常饮咖啡可降低2型糖尿病的发病率,咖啡因对糖尿病膀胱病的作用如何?有待观察。本研究通过制备糖尿病膀胱的大鼠模型,采用体外脉冲电刺激(TENS)的方法,及采用coffee or caffeine对DCP大鼠给予咖啡因水溶液(10mg·kg-1·d-1)及雀巢咖啡水溶液经口灌胃干预治疗,观察对糖尿病膀胱大鼠的排尿功能的影响,进一步通过尿动力学,细胞生物学,生物化学及分子生物学研究,从神经源性和肌源性两方面探讨TENS及coffee or caffeine对于糖尿病膀胱的作用机制及疗效。对今后临床应用TENS技术(经体表)治疗糖尿病膀胱病病变的患者及通过每日饮用适量咖啡来阻止糖尿病膀胱病病变的进一步发展,减少膀胱剩余尿(BRU),提供帮助。这将是简便而经济的方法,可取得较好的经济效益和社会效益。第一部分DCP动物模型的建立和尿动力学及病理组织学观察目的:掌握建立DCP大鼠模型的方法,为下一步干预性研究打下基础。观察DCP的尿动力学及病理学改变,初步探讨发病机制及病理演绎过程,为诊治提供依据。方法:66只SD雄性大鼠随机分为2组,即正常对照组(NC组)30只,糖尿病组(DM组) 36只、对36只8周龄的SD雄性大鼠腹腔注射STZ(60 mg/Kg)的柠檬酸缓冲液48小时后,链脲佐菌素腹腔注射诱导糖尿病大鼠模型,以连续3次测空腹血糖>12mmol·L-1为糖尿病模型建立成功。饲养60天后,进行膀胱静态压、最大膀胱容量、最大膀胱收缩压等尿动力学测定。然后行膀胱湿重、膀胱壁厚度及光镜、电镜的病理学等项观察。结果:DM组36只SD雄性大鼠48小时后,有33只大鼠空腹测尾静脉血糖>12mmol·L-1,糖尿病模型成功率达91.67%,至实验60天糖尿病大鼠总共死亡9只,存活率达72.73%。60天后,DCP鼠膀胱静态压0.95±0.10 kpa,对照组为0.60±0.03 kpa (P<0.05);DCP鼠最大膀胱容量3.49±0.40ml,而对照组为1.82±0.12ml(P<0.01);DCP鼠最大膀胱收缩压3.09±0.10 kpa,对照组为4.91±0.30 kpa(P<0.01);DCP鼠膀胱顺应性(ml/kpa)1.39±0.11 ml/kpa,对照组为0.68±0.07 ml/kpa(P<0.01);与对照组鼠相比,DCP鼠湿重,膀胱壁厚度均增加(P<0.05);DCP鼠逼尿肌边缘有空泡样变性,肌细胞核核固缩、细胞连接纤维化,成纤维细胞变性及胶原纤维排列紊乱。结论:DCP大鼠60天已有膀胱的动力学及病理学改变,提示尽管一些DM患者可无明显的排尿障碍,但客观的病理组织及尿动力学已有改变,建议对DCP患者应给予干预,控制病情的进一步恶化。第二部分TENS与非TENS对DCP干预的对照基础实验研究目的:通过制作的糖尿病膀胱大鼠模型,探讨体外电刺激的方法对糖尿病膀胱大鼠的排尿功能的影响及作用机理。方法:将SD雄性大鼠随机分为正常对照组(NC组)14只、糖尿病对照组(DM组)和糖尿病电刺激组(DM治疗组),后二组各18只,运用腹腔内注射STZ的方法制作糖尿病膀胱大鼠模型;造模成功后第10周起,DM治疗组予体外电刺激治疗,刺激参数强度31 V,密度31 Hz(二对电极分别置于膀胱体表投影区和骶尾关节上方0.5厘米处),其它二组均不予电刺激,持续治疗3周后(造模成功后第13周起)观察比较各组间尿动力学指标改变(膀胱湿重,膀胱阈容量,排尿后剩余尿量,剩余尿比值,排尿时最大膀胱压力),逼尿肌肌条收缩功能及膀胱组织光镜及超微结构的变化,通过逆转录聚合酶链式反应(RT-PCR)半定量测定背根神经节(DRG)的降钙素基因相关肽(CGRP)的mRNA含量,运用Western-blot方法检测各组大鼠膀胱壁及DRG的CGRP蛋白的表达量的变化。结果:DM治疗组大鼠与DM组大鼠相比,其膀胱收缩力增强(DM组:19.49±6.51 mmHg;DMT组:30.0±3.84 mmHg)剩余尿量比(R%)有所下降(DM组:42.83±8.71;DMT组:32.82±6.72 ),膀胱排尿阈容量减少(DM组:1.4±0.40ml;DMT组:0.93±0.26 ml ),各组间其差异均有统计学意义;组织病理学显示DM组大鼠的逼尿肌肌细胞代偿性肥大,胞内线粒体肿胀,细胞间质与胶原纤维增生明显;DM治疗组大鼠的逼尿肌肌细胞肥大明显减轻,胞内线粒体无明显肿胀,细胞间质及胶原纤维增生不明显。DM治疗组大鼠膀胱壁及背根神经节(DRG)的CGRP蛋白表达含量较DM组明显增加,DM治疗组大鼠RT-PCR结果显示其DRG的CGRP的mRNA含量也较DM组明显增多。结论:体外电刺激治疗可以①促进糖尿病膀胱大鼠膀胱壁和DRG的CGRP蛋白的合成,并有效的运输至膀胱壁发挥作用,改善膀胱充盈感觉功能,恢复排尿功能的始动因素;②修复胞浆内线粒体等细胞器的正常结构和功能,调节胞内钙离子的浓度,从而增强逼尿肌收缩力;③促进NGF因子,或神经损伤/再生长相关基因的表达,帮助完成神经的修复过程。第三部分coffee or caffeine对DCP作用的基础实验研究目的:通过建立的糖尿病大鼠动物模型,观察糖尿病大鼠膀胱的形态、功能及组织病理学变化,探讨咖啡与咖啡因对糖尿病大鼠膀胱功能障碍的影响及可能的作用机制。方法:75只SD雄性大鼠随机分为4组,即正常对照组(NC组)15只,糖尿病组(DM组)、糖尿病咖啡治疗组(Coffee组)及糖尿病咖啡因治疗组(Caffeine组)每组各20只。链脲佐菌素腹腔注射诱导糖尿病大鼠模型,以连续3次测空腹血糖>12mmol·L-1为糖尿病模型建立成功。Caffeine组大鼠给予咖啡因水溶液(10mg·kg-1·d-1)经口灌胃,Coffee组大鼠给予雀巢咖啡水溶液(286mg·kg-1·d-1)经口灌胃,其余两组以等量蒸馏水每日经口灌胃,连续7周。7周后通过尿动力学检测、离体肌条的收缩实验、放免测定及组织病理学检查等技术,测定并比较各组大鼠的膀胱湿重量、膀胱阈容量、相对排尿量(V%)、排尿后的剩余尿量、膀胱环磷酸腺苷(cAMP)含量、逼尿肌肌条的收缩力、背根神经节(DRG)的CGRP蛋白表达、膀胱组织光镜及超微结构的变化。结果:7周后相对于NC组大鼠,DM组大鼠的膀胱湿重量、膀胱阈容量及排尿后的剩余尿量明显增加(P<0.01),而V%与膀胱cAMP含量显著减少(P<0.01)。与DM组相比,Coffee组或Caffeine组糖尿病大鼠的膀胱湿重量、膀胱阈容量及排尿后的剩余尿量显著减少(P<0.05),V%与膀胱cAMP含量明显升高(P<0.05)。肌条收缩实验显示与NC组相比,DM组大鼠早期逼尿肌肌条的收缩力已明显改变(P<0.05);在Coffee组或Caffeine组,糖尿病大鼠逼尿肌肌条的收缩性明显改善(P<0.05)。组织病理学研究显示DM组大鼠的逼尿肌肌细胞代偿性肥大,胞内肌浆网代偿性增多,线粒体肿胀,细胞间质与胶原纤维增生明显;在Coffee组或Caffeine组,糖尿病大鼠的逼尿肌肌细胞肥大明显减轻,胞内肌浆网明显减少,线粒体无肿胀,细胞间质与胶原纤维增生不明显。结论:咖啡与咖啡因的治疗能改善糖尿病大鼠膀胱病变,咖啡可能通过其中的咖啡因可以增加糖尿病膀胱大鼠膀胱壁和DRG的CGRP蛋白、CGRP的mRNA的表达、增加逼尿肌收缩力及膀胱的充盈感觉功能。其机制可能是:⑴升高膀胱cAMP的含量,调节神经递质与受体作用的信号传导通路;⑵改善膀胱充盈感觉,恢复排尿反射;⑶维护胞内肌浆网、线粒体等细胞器的超微结构,调控胞内钙离子的浓度从而增强膀胱逼尿肌的收缩性;⑷增加糖尿病膀胱大鼠膀胱壁和DRG的CGRP蛋白、CGRP的mRNA的表达、增加逼尿肌收缩力及膀胱的充盈感觉功能。更多还原

【Abstract】 Diabetes mellitus is a common endocrine and metabolic disease. The diabetic neuropathy is one of the most common chronic complications of the diabetes. Diabetic cystopathy (DCP) is one of diabetic peripheral nerve pathological changes, belonging to neuropathic dysfunction of bladder (NB).Being one of the complications of urinary system caused by diabetes, its incidence occupies about 50% of all diabetic patients. Clinical researches illustrate that even if those whose blood glucose is controlled steadily, the incidence of DCP is still 25%.The definite pathogenesis of DCP is not completely known until now. Present research considers that the pathogenesis principally concludes two aspects: peripheral and autonomic nerve pathological changes, myopathic abnormal factors and so on.To seek effective methods, to prevent actively as early as possible, and to treat diabetic cystopathy pathological changes, to improve lower urinary tract symptom are important components for diabetic patients combined treatment and to raise quality of life. At present therapy of diabetic cystopathy is mainly drug therapy. Drug therapy has some effect, but side effect should not be ignored. Invasive treatment contains intermittent clean catheterization, cystostomy, transurethral resection bladder neck, and they can relieve much residual urine. Surgical invasive treatment can relieve urinary tract infection and upper urinary tract damage, but it cannot resolve fundamental problem. It may cause further infection if usage is incorrectness. How to find an effective and safe method to prevent and treat DCP pathological changes, to relieve detrusor lacking strength and much residual urine is a problem people have being explored. To prevent the DCP develops in early stage is the key to combined modality therapy and improve quality of life to diabetic patients.The reports of using Transcutaneous Electrical Nerve Stimulation(TENS) are few, and there are no researches for urodynamics and pathogenesis. Another research discovers that frequent drinking coffee can decrease the incidence of 2-diabetes.The effect of coffee for DCP is still unknown.This research is to investigate the effects of TENS on the urinary bladder voiding dysfunction in diabetic cystopathy rats by SD rat model of diabetic cystopathy rat was established. And using coffee or caffeine to perfuse diabetic cystopathy rat bladder with nasogastric tube caffeine aqueous solution(10mg·kg-1·d-1)and Nestle coffee aqueous solution. Through urodynamics, cytobiology, biochemistry and molecular biology, investigating pathogenesis and effect that TENS and coffee or caffeine have on DCP in neuropathic and myopathic aspects. It is an simple, convenient and economical way for diabetic patients to apply TENS technology and drink proper coffee or caffeine every day. It would have a great significance in economic benefit and social benefit.PartⅠⅠ:Urodynamics and pathology study in diabetic cystopathy rat Objective:To observe the abnormalities of urodynamics and histopathology in the early stage of DCP. To find a relationship between the changes in the bladder function and detrusor structure and study the onset mechanism and pathology procedure in order to provide scientific bases for early stage diagnosis and treatment.Materials and Method:66 Sprague-Dawley male rats were randomly divided into 2 groups:NC group (normal control, n=30), DM group (diabetes mellitus, n=36),. Diabetic rat model was induced by injection of streptozotocin(STZ)into peritoneal cavity. Diabetic rats were successfully accepted as diabetes by measuring fasting serum glucose concentration more than 12mmol·L-1 continually three times. In both group: the bladder resting pressure, the bladder maximum capacity, the maximum bladder pressure and bladder compliance were performed. The bladder weights were taken, bladder wall thicknesses were measured and the histopathologic structures of bladder walls were observed by optical and electric microscopy.Results: In the diabetic rats, the the maximum bladder capacity (P<0.01), compliance (P<0.05) and resting pressure (P<0.01) were increased, however the maximum bladder pressure decreased compared to the control group (P<0.01). The weight of diabetic rat bladder was increased compared to the control bladders (P<0.05). In the diabetic rats, not only the total bladder wall thickness was greater but also the muscle bundles appeared to be increased in size. In contrast to the control group, the diabetes rats showed in the detrusor cell obvious karyopyknosis, a number of compact bodies decreased and cell junction fibrosis between the detrusor cells, fibroblast degeneration and collagen fibers array disorders.Conclusions: In this study, the urodynamics in the diabetic bladders showed significant differences compared to the normal bladders. The detrusor microstructure showed to be abnormal in the early stage of diabetes. It is suggested that the bladder function is impaired obviously in the early stage of diabetes. The result of the study can be helpfully to fumble diabetic progress of bladder dysfunction in patient and therapeutic results.PartⅡ:Effects of TENS on urinary blaader voiding dysfunction in diabetic cystopathy ratsObjective:To investigate the effects of Transcutaneous Electrical Nerve Stimulation (TENS) on the urinary bladder voiding dysfunction in diabetic cystopathy rats.Methods:SD rat model of diabetic cystopathy rat was established by the injection of STZ in abdominal cavity. The rats were randomized into the DM ES group(DM with electrical stimulation,n=18), DM(diabetes mellitus,n=18) group and normal control group(n=14). TENS was performed on rats in the DM ES group in the 10th week after the establishment of diabetic rats model (stimulation parameter: intensity--31V, density—31Hz;two pairs of electrode was place on the projective area on the abdomen of the bladder respectively) , but those in the control group underwent shamoperation . After the stimulation was continuing for 3 weeks, The bladder wet weight, volume threshold for micturition, post-voided residual volume, bladder residual volume/bladder capacity (R%),the max micturition pressure, the changes of the detrusor strip contractility and bladder muscle ultrastructure were detected . The expression of CGRP mRNA in each rats’DRG was measured by RT-PCR,The expression levels of CGRP of Bladder and DRG were detected by western blot.Results: Compared with DM group , the R% and volume threshold for micturation were significantly reduced while the contractility of the detrusor muscle strip were significantly increased in DM ES group after electrical stimulation. Histopathological studies showed that the detrusor cells expressed compensatively hypertrophy, intracellular collagen fibers increased obviously and mitochondra swelled in the bladder muscle unltrastructure of diabetic rats. Electrical stimulation could reduced markedly the compensated hypertrophic cells, intercellular collagen gibers and protect mitochondria in the bladder muscle ultrastructure of DM ES group, The content of the mRNA of CGRP in DRG of DM ES group is enhanced than that in DM group. The expression levels of CGRP of Bladder and DRG in DM ES group is increased than that in DM group.Conclusion: These experiment results demonstrate that the TENS can promote the voiding in the DCP(diabetic cystopathy)rats, TENS may enhance the expression levels of CGRP of DRG and bladder in DCP, Which react in the bladder through the effective conduction, improve the bladder fullness, restore the initiate factor of the micturition. Otherwise TENS also ameliorate the configuration and function of ultrastructure, regulate the intracellular Ca2+ concentration to facilitate bladder contractility. TENS maybe accelerate the restoration of the nerver through enhace. The expression levels of NGF or the injury/regeneration related gene.PartⅢ:Effects of coffee or caffeine on DCP ratsObjective: To observe histomorphological abnormality and functional alterations of diabetic cystopathy in the streptozotocin-induced diabetic rat and explore the effects and mechanisms of caffeine and coffee on the diabetic bladder dysfunction Materials and Methods: 75 Sprague-Dawley male rats were randomly divided into 4 groups: NC group (normal control, n=15), DM group (diabetes mellitus, n=20), Coffee group (DM with coffee treatment, n=20) and Caffeine group (DM with caffeine treatment,n=20). Diabetic rat model was induced by injection of streptozotocin(STZ)into peritoneal cavity. Diabetic rats were successfully accepted as diabetes by measuring fasting serum glucose concentration more than 12mmol·L-1 continually three times. Caffeine dissolved in distilled water were administered orally (10mg/kg/day, p.o.) to diabetic rats of caffeine group for 7 weeks. According to this concentration, diabetic rats of coffee group received corresponding coffee treatment by the same method. The rest two groups were given orally the same volume of distilled water every day. After 7 weeks, the bladder wet weight,volume threshold for micturation,post-voided residual volume (PVR), V%(voided volume/bladder capacity),cAMP content of bladder, the changes of the detrusor strip contractility and bladder muscle ultrastructure were measured and compared among the groups by the methods of urodynamics, detrusor strip contractile studies, radioimmunoassay and histopathology.Results: After 7 weeks, the bladder wet weight, volume threshold for micturition and PVR were significantly higher in the diabetic rats of DM group than those in NC group(P<0.01), while V% and cAMP content of bladder were significantly lower in DM group(P<0.01). Coffee or caffeine treatment significantly reduced the bladder wet weight, bladder capacity and PVR as well as increased V% and cAMP content of bladder in the diabetic rats(P<0.05). In addition, coffee and caffeine tended to normalize the altered detrusor contractile responses to electrical field stimulation and acetylcholine in the diabetic rats. Histopathological studies showed that the detrusor cells expressed compensatively hypertrophy, intracellular sarcoplasmic reticulum and intercellular collagen fibers increased obviously and mitochondria swelled in the bladder muscle ultrastructure of diabetic rats. Coffee or caffeine treatments reduced significantly the compensated hypertrophic cells, intracellular sarcoplasmic reticulum and intercellular collagen fibers and protect mitochondria in the bladder muscle ultrastructure of diabetic rats.Conclusions: These results indicate that the bladder functions were impaired obviously in the early stage of diabetic rats. Caffeine and coffee may have beneficial effects on bladder dysfunction in the early stage of diabetic rats by up-regulation of cAMP content in the lower urinary tract mediating cell-signal transduction pathways, improvement of the bladder fullness sensation, recovery of the micturition reflex and protection of bladder ultrastructure regulating intracellular Ca2+ concentration to potentiate bladder contractility.更多还原