【作者】 鲁湘鄂；

【导师】 吴清和；

【作者基本信息】 广州中医药大学 ， 中药学， 2012， 博士

【摘要】 在临床上,膀胱逼尿肌功能障碍引起的尿频、尿急、尿失禁和遗尿极为常见。而逼尿肌收缩性、兴奋性和顺应性是影响膀胱功能的主要因素。缩泉丸出自《妇人良方》,由乌药、益智仁、山药组成。主治尿多,尿频,遗尿。具有温化肾气,缩尿止遗的功效,其功效可概括为“补肾缩尿”。研究已经证实,直接介导膀胱逼尿肌收缩的主要受体是M3R,介导膀胱逼尿肌松弛的主要受体是β3-AR。目的：以调节膀胱逼尿肌细胞收缩功能的M3R及其介导的信号转导通路和调节膀胱逼尿肌细胞舒张功能的p3-AR及其介导的信号转导通路为出发点,立足于逼尿肌功能异常与神经源性的相关性,以膀胱逼尿肌细胞为研究对象,阐明缩泉丸“缩尿”功效与现代医学调节逼尿肌舒缩功能的内在联系。方法：以自然衰老大鼠为“肾虚多尿”模型。取筛选合格的自然衰老大鼠(15月龄)70只,青年大鼠(3月龄)14只。设置组别：青年大鼠为正常对照组,14只,自然衰老大鼠分为模型对照组、金匮肾气丸组、缩泉丸低、中、高剂量组,14只/组。金匮肾气丸组的给药剂量为1.08g/kg/d;缩泉丸低、中、高剂量分别为0.293g/kg/d、0.585g/kg/d、1.170g/kg/d。采用酶消化与组织块相结合的方法原代培养自然衰老大鼠膀胱逼尿肌细胞；采用RT-PCR方法测定膀胱逼尿肌细胞M3R和β3-AR mRNA的表达；采用western blotting方法测定膀胱逼尿肌细胞M3R和β3-AR蛋白的表达；采用酶联免疫分析试剂盒测定膀胱逼尿肌细胞IP3含量；采用激光扫描共聚焦显微镜测定膀胱逼尿肌细胞Ca2+和CaM浓度。结果：1、缩泉丸减少自然衰老大鼠的尿量,提示缩泉丸有“缩尿”的作用；2、成功地原代培养出自然衰老大鼠膀胱逼尿肌细胞；3、缩泉丸能使自然衰老大鼠逼尿肌细胞M3R mRNA表达降低,β3-AR mRNA表达增加；4、缩泉丸能使自然衰老大鼠逼尿肌细胞M3R蛋白表达降低,β3-AR蛋白表达增加；5、缩泉丸能使自然衰老大鼠逼尿肌细胞的IP3的含量降低；6、缩泉丸使H-89阻断β3-AR介导的信号通路的自然衰老大鼠逼尿肌细胞PKA蛋白表达增加；7、缩泉丸使自然衰老大鼠逼尿肌细胞Ca2+和CaM浓度均降低。结论：缩泉丸下调M3R及其介导的信号转导通路和上调β3-AR及其介导的信号转导通路,从而维持了二者的动态平衡,缩泉丸能使自然衰老大鼠逼尿肌细胞Ca22+和CaM浓度均下调,降低逼尿肌的收缩力和兴奋性,减少逼尿肌异常收缩频率,保证了膀胱正常的生理功能。将缩泉丸“缩尿”理论结合神经源性来探讨缩泉丸作用的多靶性,为缩泉丸治疗肾虚多尿提供了确切的分子药理学基础,为中医“补肾缩尿”的理论给予新的诠释。更多还原

【Abstract】 Frequent urination,urgency of urination,urinary incontinence and enuresis caused by Bladder detrusor dysfunction are very common in the clinic. The main factor affecting the bladder function is contractility, excitability and compliance of detrusor.SQW is reeorded in Chinese medicine book named Furenliangfang. SQW consists of three herb, Radix Linderae, Radix dioscoreae and Alinia oxyphylla Miqfrom.The function of SQW is polyuria,urinary frequency and enuresis,and is to warm kidney,stop Polyurial and hold exeessive urination.Its effect can be summarized as" tonifying kidney and shrinkaging urinary". Studies have confirmed that the bladder detrusor contraction is directly mediated by M3muscarinic acetylcholine receptor (M3R) and the detrusor relaxation is directly mediated by β3-adrenoceptor(β3-AR).Objective:This study was intended to regard β3-AR regulating bladder detrusor relaxation and its signal transduction pathway and regard M3R regulating bladder detrusor contraction and its signal transduction pathway as cut-in point of the research. This study was based on the dependablity of dysfunction and neurogenic of the bladder detrusor and the bladder detrusor cells was regarded as the research object. The research was elucidated the immanent connection of" reducing urination " effect and regulating diastolic and systolic function on modern medical.Methods:Natural aging rats were used as " kidney deficiency and urinary " model. After preliminary screening,70natural aging rats were15months old,14young rats were3months old. The Setting groups were as follows:young rats were regarded as normal control group and natural aging rats were divided into model control group, Jinkui Sheni Pill,SQW low dose group, SQW middle dose group and SQW high dose group on average. The dosage of Jinkui Sheni Pill was1.08g/kg/d. The dosage of Suoquan Wan low, middle and high dose group was respectively0.293g/kg/d,0.585g/kg/d,1.170g/kg/d. primary culture of natural senescent rats detrusor cell was cultured by the combination method of enzymatic digestion and tissue. M3R and β3-AR mRNA expression of urinary bladder detrusor cells were determined by RT-PCR.M3R and P3-AR protein expression of urinary bladder detrusor cells were determined by Western blotting. IP3content of urinary bladder detrusor cells was determined by enzyme-linked immunoassay reagent kit. Ca-and CaM concentration of urinary bladder detrusor cells were determined by confocal laser scanning microscopeResults:1. Suoquan Wan reduced urine volume of natural aging rat.This prompt " reducing urination " effect of SQW.2. Bladder detrusor cells of Natural aging rat were easily cultured.3. SQW could make M3R mRNA expression of detrusor muscle cells of the natural aging rats decreased,and β3-AR mRNA expression of detrusor muscle cells of the natural aging rats increased.4. SQW could make M3R protein expression of detrusor muscle cells of the natural aging rats decreased,and β3-AR protein expression of detrusor muscle cells of the natural aging rats5. SQW could make IP3content of detrusor muscle cells of the natural aging rats decreased.6. SQW could make PKA protcin content of blocking signal pathway by H-89increased.7. SQW could make Ca2+and CaM concentrations of detrusor muscle cells of the natural aging rats decreased.SQW could make M3R and its mediated signal transduction pathway down-regulated,makeβ3-AR and its mediated signal transduction pathway up-regulated and make the Ca2+and CaM concentrations of rat detrusor cells of natural aging down-regulated. SQW reduced detrusor contractility and abnormalities contraction frequency, guaranteed the normal physiological function of bladder. The " reducing urination " effect was combined with neurogenic to discuss multiple target of SQW.It provided exact molecular pharmacology basis for SQW treating "kidney deficiency and urinary " and gived "tonifying kidney and shrinkaging urinary" theory to a new interpretation更多还原