从神经-ICC-平滑肌网络探讨补肾缩尿法对膀胱过度活动症的作用机制

发布时间：2018-02-22 22:48

本文关键词： 缩泉丸联用肾气丸补肾缩尿法膀胱过度活动症豚鼠膀胱神经-ICC-平滑肌网络　出处：《广州中医药大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的：膀胱过度活动症(Overactive Bladder,OAB)作为一种以下尿路病理和尿急症状为特征的病症,其明显对患者的日常生活和社会活动造成极大的干扰,已经成为困扰人们的一大疾病。神经-ICC-平滑肌网络作为新近认为是OAB的其中一个致病原因,日益受到研究人员关注的研究热点。缩泉丸的功效为补肾缩尿,固脱止遗。肾气丸功效为温补肾阳,振奋阳气,缩泉丸与肾气丸合用在治疗OAB上有良好的疗效。本课题以神经-ICC-平滑肌网络为切入点,研究补肾缩尿法对OAB的作用机制。方法：本研究以膀胱出口梗阻模型作为OAB的研究模型。将豚鼠分为正常组、模型组、阳性组、缩泉丸组、肾气丸组和缩泉丸联用肾气丸组六个组。分成三部分1、补肾缩尿法对豚鼠OAB的治疗作用：测各组的排尿次数、排尿量、尿动力参数和离体肌条功能参数。2、研究补肾缩尿法对OAB豚鼠膀胱神经-ICC-平滑肌网络结构的影响：通过组织免疫荧光法观察膀胱ICC、乙酰胆碱能神经(VAChT)、P物质肽能神经的相对含量变化,以及透射电镜观察整体膀胱神经-ICC-平滑肌网络结构；3、研究补肾缩尿法对OAB豚鼠膀胱神经-ICC-平滑肌网络信号传导的影响：通过组织免疫荧光双标观察ICC与M3、P2X5受体亚型的共表达情况,观察补肾缩尿法是否作用于ICC上的M3和P2X5受体,以及采用荧光定量PCR法、westernblotting法检测ICC-平滑肌网络缝隙连接蛋白Cx43mRNA及蛋白的表达。结果：1、模型组能显著增加豚鼠排尿次数,增加膀胱漏尿点压力(BLPP),最大膀胱压(MVP)及残余尿量(PVR)的作用(P0.05)；模型组显著提高了膀胱肌条对激动剂的刺激敏感性,对由于KCL激动剂引起的收缩和Carbachol引起的收缩有显著性提高(P0.05)；模型组ICC相对表达量显著升高,而肽能神经的相对表达量显著降低(P0.05),模型组膀胱神经-ICC-平滑肌网络之间的连接更为紧密；ICC上神经受体M3和P2X5表达量在造模后显著升高(P0.05),缝隙连接蛋白Cx43的mRNA和蛋白表达量显著升高(P0.01)。2、补肾缩尿法对豚鼠OAB的治疗作用：用托特罗定、缩泉丸或缩泉丸联用肾气丸组治疗能减少OAB模型豚鼠的排尿次数(P0.05),托特罗定、缩泉丸、肾气丸以及缩泉丸联用肾气丸组有不同程度降低BLPP、MVP及PVR的作用(P0.05)。托特罗定、缩泉丸以及缩泉丸联用肾气丸组均有不同程度显著性降低由于KCL激动剂所引起的肌条收缩作用(P0.05),并在Carbachol不同浓度影响下的收缩作用均有显著性降低(P0.05)。3、补肾缩尿法对OAB豚鼠膀胱神经-ICC-平滑肌网络结构的影响：免疫荧光结果显示,给予缩泉丸、肾气丸和缩泉丸联用肾气丸有不同程度降低ICC细胞和提高神经纤维P物质的相对表达量,其中缩泉丸联用肾气丸效果最为显著(P0.05,P0.01)。透射电镜图显示,分别给予托特罗定,缩泉丸,肾气丸或补肾缩尿法治疗后,均有不同程度改善神经-ICC-平滑肌结构间的联系,联系减少。4、补肾缩尿法对OAB豚鼠膀胱神经-ICC-平滑肌网络信号传导的影响：对神经受体与ICC的免疫荧光双标结果显示,补肾缩尿法治疗后,ICC细胞上的M3神经受体表达显著下降(P0.05)；缩泉丸联用肾气丸干预后,膀胱缝隙连接蛋白Cx43mRNA和蛋白表达量呈极显著下降(P0.01),证明缩泉丸联用肾气丸能够通过减少冲动信号的传递而达到调节膀胱功能。结论：1、模型组能增加排尿次数,伴有平滑肌不稳定收缩,并对激动剂的敏感性伴有显著性升高,代表造模成功。其中膀胱ICC数量升高,代表OAB的发生与ICC数量变化有关。2、缩泉丸联用肾气丸组明显改善OAB豚鼠排尿次数,能够减少膀胱漏尿点压力(BLPP)、最大膀胱压(MVP)及残余尿量(PVR),改善膀胱肌条张力活动,初步证明补肾缩尿法具有改善OAB膀胱病理状态的作用。3、模型组ICC数量明显增加,神经纤维数量明显增加,透射电镜结果显示神经-ICC-平滑肌网络联系更加紧密。经缩泉丸联用肾气丸组治疗能够改善膀胱神经-ICC-平滑肌网络的联系,改善去神经化的病理状态,预示给药后药物能通过降低ICC细胞和改变神经纤维数量从而起到补肾缩尿的作用。4、缩泉丸联用肾气丸组能减少神经受体的数量,减低缝隙连接蛋白的表达量,起到减少OAB豚鼠膀胱神经-ICC-平滑肌网络信号传导。说明补肾缩尿法通过改善神经-ICC-平滑肌网络之间的联系起到补肾缩尿的作用。
[Abstract]:Objective: overactive bladder (Overactive Bladder, OAB) as a kind of urinary tract and bladder symptoms following pathological condition characterized by its obvious, on the patient's daily life and social activities caused great disturbance, has become a major disease of people. -ICC- neural network as a newly smooth muscle is considered a cause OAB which has been the research focus of attention of researchers. The effect of suoquanwan for nourishing kidney and reducing urine gutuo bushenzhiyi. Efficacy of Shenqi Pill for warming Yang, activating Yang Qi, suoquanwan and Shenqi Pill combination has good curative effect in the treatment of OAB. The -ICC- neural network for smooth muscle the starting point, study the mechanism of nourishing kidney and reducing urine of OAB. Methods: in this study, bladder outlet obstruction model as the research model of OAB. The guinea pigs were divided into normal group, model group, positive group, suoquanwan group, Shenqi Pill group and Suoquan Shenqi Pill group six group combination pills. Divided into three parts 1, the treatment effect of tonifying kidney and reducing urine of guinea pig OAB: measured the frequency of urination, urine volume, urine dynamic parameters and function parameters from.2 body muscle, effects of nourishing kidney and reducing urine of guinea pig bladder smooth muscle -ICC- OAB neural network structure. By immunofluorescence observation of bladder ICC, cholinergic nerve (VAChT), changes in the relative content of P substance peptidergic nerve, and transmission electron microscopy. The whole bladder smooth muscle -ICC- neural network structure; 3, effect of nourishing kidney and reducing urine of guinea pig bladder smooth muscle -ICC- OAB neural network signal transduction through tissue immunofluorescence observation ICC and M3 co expression of P2X5 receptor subtypes, M3 receptor and P2X5 observation of nourishing kidney and reducing urine method is applied on the ICC, and the fluorescent quantitative PCR method was used to detect ICC- smooth muscle westernblotting joint network The expression of gap junction protein Cx43mRNA and protein. Results: 1, the model group significantly increased the frequency of urination in guinea pigs, increase bladder leak point pressure (BLPP), maximum bladder pressure (MVP) and residual urine volume (PVR) effect (P0.05); model group significantly increased the bladder muscle strips of excited stimulation sensitivity agent, can significantly improve the induced by KCL agonist induced contraction and contraction of Carbachol (P0.05); model group ICC expression was significantly increased, while the relative expression of peptidergic nerve decreased significantly (P0.05), the connection between the model group of bladder smooth muscle -ICC- neural network more closely; ICC nerve receptors M3 and P2X5 expression increased significantly in the model (P0.05), mRNA and protein expression of gap junction protein Cx43 was significantly higher (P0.01).2, the treatment effect of tonifying kidney and reducing urine of guinea pig OAB: Todd Luoding, suoquanwan or Suoquan pill combined with Shenqi Pill group Treatment can reduce the frequency of urination of guinea pig OAB model (P0.05), Todd Luoding, suoquanwan, Shenqiwan and combined with Shenqi Pill group to different degrees. BLPP suoquanwan, MVP and the role of PVR (P0.05). Luoding Todd, suoquanwan and Suoquan pill combined with Shenqi Pill group had different degree due to significantly reduced muscle KCL agonist induced contraction (P0.05), and the contraction had significant effects on Carbachol under different concentrations decreased (P0.05).3, effects of nourishing kidney and reducing urine of guinea pig bladder smooth muscle -ICC- OAB neural network structure: the immunofluorescence results showed that given Suoquan pill, Qi pill and Suoquan pill combined with Shenqi Pill with different degrees of reduction of ICC cells and increase the relative expression of nerve fiber P amount of substance, which Suoquan pill combined with Shenqi Pill was the most significant (P0.05, P0.01). Transmission electron microscope showed that were given Luoding Todd, suoquanwan, kidney Gas pill or method of nourishing kidney and reducing urination after treatment were improved -ICC- nerve smooth muscle structure between the contact to reduce.4, effects of nourishing kidney and reducing urine of guinea pig bladder smooth muscle -ICC- OAB neural network signal transduction: immunofluorescence staining of nerve receptor and ICC show that the method of nourishing kidney and reducing urine after the treatment, the expression of M3 nerve receptors in ICC cells decreased significantly (P0.05); Suoquan pill combined with Shenqi Pill intervention, bladder gap junction protein Cx43mRNA and protein expression was significantly decreased (P0.01), proof of Suoquan pill combined with Shenqi Pill can transfer by reducing the impulse signal and to adjust the bladder function. Conclusion: 1 the model group, can increase the frequency of urination, accompanied by unstable contraction of smooth muscle, and sensitivity to agonist was associated with a significant increase, on behalf of the successful establishment of the model. The number of ICC increased the number of bladder, occurrence and change of ICC on behalf of OAB are .2, Suoquan pill combined with Shenqi Pill group significantly improved OAB guinea pig urination, can reduce bladder leak point pressure (BLPP), maximum bladder pressure (MVP) and residual urine volume (PVR), improve the tension activity of bladder muscle strips, preliminary proof method has the effect of nourishing kidney and reducing urine.3 improve the pathological state of bladder OAB the number of ICC in the model group increased significantly, the number of nerve fibers increased significantly. The TEM results showed that the -ICC- neural network is more closely linked. The smooth muscle of Suoquan pill combined with Shenqi Pill group therapy can improve bladder smooth muscle -ICC- neural network, improve the pathological state of denervation, that after administration of drugs by reducing effect the number of.4 ICC cells and the change of nerve fibers to nourishing kidney and reducing urine, Suoquan pill combined with Shenqi Pill group can reduce the number of neural receptors, the expression of gap junction protein decreased, to reduce OAB in guinea pig bladder nerve -ICC- The smooth muscle network signal transduction. It shows that the tonifying kidney and urination method plays the role of tonifying kidney and reducing urine by improving the connection between the neural -ICC- smooth muscle network.

【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R277.5

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