【作者】 杨玻；

【导师】 齐清会；

【作者基本信息】 大连医科大学 ， 中西医结合外科学， 2010， 博士

【摘要】 背景：慢性前列腺炎(Chronic prostatitis, CP)是泌尿外科常见病、多发病,其病因、病理改变和临床症状复杂多样,发病机制不明,其中自身免疫反应可能起着重要的作用。排尿功能障碍是CP主要临床表现之一,目前认为功能性后尿道梗阻是CP排尿功能障碍的主要原因。梗阻可诱发逼尿肌无抑制收缩,形成不稳定膀胱。部分CP患者有膀胱的尿动力学异常改变,但对其产生的机制相关的研究相对较少。膀胱逼尿肌中存在的ICC样细胞与神经末梢关系密切,具有起搏功能,研究发现不稳定膀胱中ICC样细胞的数量及存在形式都发生了改变。膀胱功能异常是排尿功能障碍的常见原因,那么慢性前列腺炎的膀胱ICC样细胞的数量及存在形式都发生了怎样的改变,以及这种改变与膀胱功能异常之间的关系如何?值得深入研究。青藤碱(SIN)有抗炎和免疫抑制以及对胃肠道平滑肌的抑制作用,而应用于临床；针灸治疗慢性前列腺炎及抑制膀胱活动亢进的作用也有明确报道。但其作用机理鲜有报道。目的：观察慢性非细菌性前列腺炎大鼠模型膀胱功能及膀胱ICC样细胞网络的变化,研究中药青藤碱和针刺治疗对慢性前列腺炎及膀胱功能的影响,探讨青藤碱和针刺治疗慢性前列腺炎的机理。为慢性前列腺炎及其膀胱功能障碍的治疗提供新的思路。方法：采用同种系SD大鼠前列腺蛋白提纯液,辅以双重免疫佐剂方法,建立实验性慢性非细菌性前列腺炎(CAP)大鼠动物模型。将动物随机分为模型对照组、针刺组、青藤碱大、中、小剂量组五组。青藤碱大、中、小剂量组分别为30mg/kg、20 mg/kg和10 mg/kg体重,腹腔注射,每日一次,连续14天。针刺组电针中膂俞、会阳,电针治疗参数：输出电压4～6 V,疏波4 Hz,密波20 Hz,留针20 min。1次/d,共治疗10d。治疗后尿流动力学检测描记各组膀胱内压力图,取前列腺组织,观察其病理改变,逼尿肌组织铺片,免疫荧光检测c-kit阳性细胞的表达。用用双抗体夹心.酶联免疫吸附试验(ABC-ELISA)法测定前列腺组织匀浆IL-1β、PGE2和β-EP的含量。RT-PCR检测膀胱逼尿肌中NGFmRNA的表达。结果：与正常大鼠相比,模型组动物充盈性膀胱压力测定显示膀胱最大容量和最大逼尿肌压明显减小,残余尿量明显增加,70%的大鼠在膀胱充盈期出现程度不等的逼尿肌不稳定性收缩。模型组前列腺病理符合慢性前列腺炎改变,证实CAP可以引起膀胱功能的改变,为前列腺炎膀胱功能异常提供了功能学上的依据。但模型组慢性前列腺炎各病理分级中不稳定膀胱比率无明显差别。与正常对照组相比,模型组Kit阳性的ICC样细胞数量增多,积分光密度值(IOD)降低,部分ICC样细胞排列紊乱,细胞间的联系散乱与模型组相比,针刺会阳、中膂俞和青藤碱治疗后,发生逼尿肌不稳定收缩的动物数量明显减少,程度减轻。青藤碱大剂量组和针刺组大鼠前列腺上皮组织恢复正常,管壁结构完整,间质水肿消失。青藤碱中剂量组和小剂量组对实验大鼠前列腺组织结构的破坏虽然也有一定程度的改善,但作用较轻。与模型组相比,不同剂量青藤碱治疗组随青藤碱使用剂量增加,ICC样细胞数量逐渐减少,IOD逐渐增高,以青藤碱大剂量组最接近正常对照组。模型组NGF mRNA表达明显升高,各青藤碱治疗组随青藤碱剂量增加NGF mRNA表达依次降低,针刺组和青藤碱大剂量组NGF mRNA表达接近正常组。各组逼尿肌组织NGF的改变与ICC样细胞的变化呈现一致性。青藤碱大、中、小剂量组及针刺组大鼠前列腺组织内PGE2及IL-1β的水平较模型组明显降低；β-EP的水平较模型组明显升高。青藤碱大剂量组、针刺组大鼠前列腺组织匀浆中IL-1β含量与正常组比较,差异无统计学意义(P>0.05)结论：采用自身免疫反应所造成的CAP动物模型,其病理改变与临床的病理改变较为接近,是一种较为理想的的造模方式。慢性前列腺炎模型大鼠可以出现以逼尿肌不稳定收缩为主的膀胱功能异常,以及膀胱ICC样细胞分布和数量的异常。膀胱ICC样细胞结构和功能的变化可能与CP膀胱功能异常的产生有关。盐酸青藤碱和针刺会阳、中膂俞能够降低大鼠前列腺组织内PGE2及IL-1β的水平,升高p-EP的水平,可减轻前列腺组织的炎症反应,改善前列腺组织结构的破坏,对前列腺组织起到一定的保护作用。针刺会阳、中膂俞和青藤碱对前列腺组织的保护作用可以影响膀胱ICC样细胞的数量和分布,减轻继发的膀胱的肌源性损害,从而调节膀胱功能。针刺和青藤碱对慢性前列腺炎膀胱NGF产生抑制作用,可以从某种程度上说明膀胱ICC样细胞与NGF存在交互作用,提示,针刺和青藤碱可能通过免疫抑制和对细胞因子和神经递质的调节等作用参与到膀胱ICC样细胞网络的调节。更多还原

【Abstract】 Background:Chronic non-bacterial prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a common disease of male reproductive system. The etiology and the pathogenesis of CP are not yet clear so far. Because of the high incidence of the disease, lack of typical symptoms, and difficulty to treat, CP seriously impairs the quality of life in patients. Recently studies have shown that CP is the result of a variety of factors. Autoimmune reactions play an important role in the pathogenesis which is mediated by inflammatory cytokines. Pain, voiding dysfunction and sexual dysfunction are three main manifestations of CP. Many investigations ascribed the voiding dysfunction to the functional obstruction at posterior urethra which evokes non-inhibitive contraction and eventually leads to unstable bladder. However, among some patients without functional urethral obstruction, symptoms and abnormal urodynamic findings are also present.Interstitial cells of Cajal (ICC) in the bladder act as pacemaker and have strong relationship with nerve terminal. The increase of ICC-like cells in the detrusor, which maybe lead to an enhanced spontaneous contraction of detrusor, and may be one of the important mechanisms of detrusor instability (DI). The abnormal function of bladder was the main reason for the voiding dysfunction. The question is:are there any relationship with the alteration of bladder ICC-like cells in the association between voiding dysfunction and CP? To the best of our knowledge, there were very few studies addressing this question.Sinomenine was widely used in clinical practice because of its effects of anti-inflammatory, immunosuppression and inhibitory action to gastro-intestinal smooth muscle. It was recognized that acupuncture on relative points had therapeutic effects on CP symptoms and bladder hyperactivity. As such, elucidation of the mechanism at myogenic level of detrusor of bladder will facilitate treatment of CP and the concomitant bladder dysfunction.Objective:To observe the alteration of bladder function and its ICC-like cells in SD rat model of experimental autoimmune prostatitis (EAP), and to investigate the effects of Sinomenine and acupuncture at Zhonglushu and Huiyang point on morphological peculiarities of experimental rats prostate tissue and the concomitant bladder dysfunction, and evaluated their efficacy.Method:The model of experimental autoimmune prostatitis (EAP) rat was made by DPT vaccine in peritoneal injection and rat prostatein purification of FCA emulsion multiple intra-dermal injections for 45 days. Cystometries were performed in conscious rats after suprapubic bladder puncture. The bladder capacity, pressure and postvoiding volume were measured and the incidence of detrusor instability was studied. Then prostate glands were obtained to investigate the histomorphological changes. Detrusor tissue pieces were obtained for detecting c-kit positive cells by immunofluorescent staining.Fifty SD rats were randomly divided into a control group, a model group, an acupuncture group and the groups of low, medium and high dose of Sinomenine. The low, medium and high dose group was given dose of 10, 20,30mg/kg, respectively, by intraperitoneal injection. The acupuncture group was electroacupunctured at Huiyang (BL 35) and Zhonglushu (BL 29) with output voltage of 4～6V, dense wave of 20 Hz and distant wave of 4 Hz. The above groups were subsequently treated for 14 days, qd. Finally, cystometries, histomorphological observation of prostate and detection of c-kit positive cells in bladder were performed. The bladder detursor tissues were harvested for a reverse transcription polymerase chain reaction (RT-PCR) exam of NGFmRNA.The contents of IL-1β, PGE2 and β-endorphin in prostate-homogenate were detected by enzyme linked immunosorbent assay.Results:The incidence of DI developed in model group was 70%. Bladder pressure and capacity decreased from (27.3±1.87) cmH2O and (0.57±0.06) mL in control rats to (22.8±2.03)cmH2O and (0.39±0.05)mL in model rats, respectively (P< 0.05). The postvoiding volume increased from (0.11±0.04)mL to (0.33±0.06)mL. The model group showed chronic inflammation in prostate glandular tissue. The above results indicated that the model was built successfully and was a candidate for further study on bladder dysfunction secondary to CP.Compared with control group, model group showed considerably increase in number of ICC-like cells and decrease of integral optical density value (IOD) in the detrusor. The ICC-like cells were arranged disordered, and the intercellular connection was scattered in model group.After acupuncture and administration of Sinomenine, DI ratio of model group, acupuncture group, Sinomenine high dose group, medium dose group and low dose group was 60%,10%,20%,40%, and 50%, respectively. Compared with that of the model group,the bladder capacity and maximum detrusor pressure increased,and the residual volume decreased significantly in the treatment group.In the high dose group of Sinomenine and acupuncture group, the epithelial tissues recover, the architecture of epithelial tissues were integrated, and the interstitial edemas disappeared. In the mid and low dose group, the damage of prostate gland tissues recovered slightly. According to classified statistical analysis, the differences among each group of the Sinomenine and the acupuncture group had statistical significance.Compared with the model group, the number of ICC-like cells (c-kit positive cells) decreased significantly and ICC-like cells were arranged more orderly in the Sinomenine and acupuncture groups. The expression levels of NGF in high dose and mid dose group of Sinomenine and in acupuncture group were significantly decreased compared with that in model group, the alteration of expression levels of NGF and ICC-like cells had positive correlationCompared with the model group, the level of PGE2 and IL-1βof prostate glandular tissues reduced in both Sinomenine and acupuncture groups, whereasβ-EP increased obviously.Conclusion:Using the way of autoimmune reaction to induce the CP animal model, the change in pathology of the animal’s prostate tissue was most similar to that of patients, and pathogenesis is relatively consistent with patients. It was a satisfactory way to induce CP animal model.The CP animal model was present with bladder dysfunction of DI and alteration of ICC like cells in detrusor. The alteration of ICC-like cells in number and distribution was associated with the voiding dysfunction of CP rat model.Sinomenine and acupuncture at Zhonglushu and Huiyang point can remarkably lower down the levels of PGE2 and IL-1β, increase theβ-EP level in rats with nonbacterial chronic prostatitis, reduce or eliminat inflammation of rat prostate tissue, and improve its organizational structure damage. They had an anti-fibrosis effect and play an important role in protecting prostate issue.The results of this study were suggested that there would be interaction between ICC-like cells and NGF in detrusor, and the protective effect of Sinomenine and acupuncture on prostate tissue would influence the quantity and distribution of bladder ICC-like cells directly or by means of immunosuppression and regulation of neurotransmitters.更多还原