【作者】 张丽；

【导师】 金杭美；

【作者基本信息】 浙江大学 ， 妇产科学， 2007， 硕士

【摘要】 背景压力性尿失禁（stress urinary incontinence，SUI）是一种较为常见的中老年女性疾病，常和盆底脏器脱垂（pelvic organ prolapse，POP）相伴发生，表现为腹压增大时尿液经尿道的不自主流出，是老年化国家最常见的疾病之一。在美国，女性尿失禁发病率约为40％，每年有超过30万的妇女要求手术治疗，其中三分之一的患者需要再次手术。压力性尿失禁可严重影响患者的生活质量，近年来对其研究呈逐年增多趋势。女性压力性尿失禁的病因目前尚不十分清楚，主要由盆底解剖结构改变以及内在的括约肌功能不良引起。研究发现，其发病可能与年龄、婚育、绝经后、阴道分娩及妇科手术等因素有关，此外，肥胖、便秘、慢性咳嗽、家族史、吸烟史以及体内雌激素水平等也可能影响其发病。同样，在压力性尿失禁的诊断以及术前、术后评估目前亦无可靠的指标。因此，对其发病机制的进一步深入研究以期寻找可能的阻断途径以及寻找更为有效的疾病诊断和监测指标无疑具有非常重要的意义。从结构上分析，女性的低位泌尿生殖系统是由盆内肌肉、韧带以及骨性骨盆所支撑，完整的、有功能的结缔组织是支撑和固定其解剖位置、抵御腹腔压力的重要因素。其中的Ⅰ型和Ⅱ型肌纤维以及某些胶原是维持盆底收缩力的主要成分。研究发现，和正常人比较，73％SUI患者盆底肌肉组织存在收缩功能不良。Hale等人也证实SUI患者盆底及细胞有不同程度的萎缩变性，肌细胞凋亡。Boreham等发现POP患者盆底组织各型胶原比例改变，含量下降，弹性蛋白成分下降。另外，和SUI相关的一些危险因素如阴道分娩、产伤等亦能造成盆底结缔组织不同程度的损伤。包括阴道壁在内的尿道周围组织与膀胱尿道紧密相连，它们作为膀胱尿道的支持结构，既相互联系又相互影响。有研究表明，阴道前壁以及尿道周围组织对机械压力较为敏感因此能较准确地反映盆底结缔组织的支持力。而近年来大量实验表明，钙蛋白酶（calpain）是人体组织中普遍存在的中性蛋白酶，和各类细胞骨架蛋白的降解密切相关，在肌纤维、胶原以及其他细胞外基质的降解过程中也发挥重要作用，多种疾病的发生和钙蛋白酶／钙蛋白酶抑素表达失衡有关，因此猜测calpain可能在SUI的发病过程中起作用。Calpain是一种Ca²⁺依赖的中性蛋白酶，按存在的部位可分为组织特异性calpain和非组织特异性calpain，前者包括calpain-1、2、5、7、10、13和15，后者包括calpain-3、6、8、9、11和12。依据其激活时需要的Ca²⁺浓度，钙蛋白酶又可分为calpain-1（u-calpain）和calpain-2（m-calpain），calpain-1激活所需要的Ca²⁺浓度为5-50umol／L，而calpain-2激活所需要的Ca²⁺浓度为0.2-1.0mmol／L。calpain-1和calpain-2都由80KD的大亚基和30KD的小亚基组成，其中大亚基为活性中心，小亚基则具调节功能。目前已发现50多种钙蛋白酶抑制剂，包括胞内胞外蛋白以及一些对半胱胺酸酶有抑制作用的药物碘醋酸盐等，其中钙蛋白酶抑素（calpastatin）是胞内专一性抑制钙蛋白酶活性的蛋白质，它可以识别钙蛋白酶与钙结合引起的构象变化并与之特异结合，从而抑制蛋白酶活性保证钙蛋白酶对底物只进行局部的特定位点的水解。在生理状态下，calpain具有多种作用，如修饰受体蛋白、改变细胞骨架成分和介导胞内信号传导等。但calpain被病理性激活就会引起疾病的发生。已证实，肢带型肌营养不良，二型糖尿病，阿耳茨海默（氏）病，类风湿性关节炎等众多疾病的发生和钙蛋白酶的异常改变存在密切关系，而calpain抑制剂的应用也为相关疾病发生和疾病进展过程的阻断提供了可能。本研究拟通过分析压力性尿失禁患者尿道周围组织中calpain及calpastatin的表达情况，探讨calpain／calpastatin在压力性尿失禁发病过程中的意义，分析可能相关的高危因素，预测复发风险，对指导临床实践有重要意义。方法与结果方法：采用RT-PCR法和免疫印迹（western blotting）法对31例正常尿道周围组织（对照组）、39例SUI患者（SUI组）尿道周围组织中calpain-1、calpain-2和calpastatin的mRNA及蛋白含量进行检测。SUI诊断依据国际控尿协会标准，且所有研究对象近3月内未服用过激素类药物。对照组入选条件：无尿失禁症状，妇科检查无阴道壁膨出或子宫脱垂，近3月内未服过用激素类药物，术后病理检查证实为非子宫内膜异位症等雌激素相关疾病。结果：（1）SUI组和对照组尿道周围组织中calpain-1的表达SUI组和对照组尿道周围组织中均有calpain-1 mRNA和蛋白的表达，其中calpain-1 mRNA的表达水平两组分别为0.79±0.14和0.62±0.07，蛋白表达水平两组分别为0.72±0.07和0.69±0.09，两组相比较均无显著性差异（p＞0.05，p＞0.05）。（2）SUI组和对照组尿道周围组织中calpain-2的表达SUI患者和非SUI患者尿道周围组织中均有calpain-2 mRNA和蛋白表达，但表达强度存在差异。SUI组calpain-2 mRNA和蛋白表达水平为1.36±0.06和0.75±0.07，分别高于对照组的1.16±0.04和0.56±0.06，两组间比较有统计学差异（p＜0.05，p＜0.05）。（3）SUI组和对照组尿道周围组织中calpastatin的表达SUI组和对照组尿道周围组织中均有calpastatin mRNA和蛋白表达，但表达强度存在差异。SUI组calpastatin mRNA表达水平为0.85±0.07，高于对照组的0.62±0.06（p＜0.05），而蛋白表达水平却为0.72±0.06，显著低于对照组的1.84±0.09（p＜0.01）。结论1、SUI患者尿道周围组织中calpain-2 mRNA和蛋白表达水平较对照组增高。2、SUI患者尿道周围组织中capastatin mRNA表达水平较对照组增高，而蛋白表达水平却显著降低。3、Calpain／calpastatin表达失衡可能参与压力性尿失禁的发生，calpastatin可能在阻断压力性尿失禁的发生和发展中起作用，推测钙蛋白酶抑制剂有潜在的治疗作用。更多还原

【Abstract】 IntroductionFemale stress urinary incontinence （SUI） is a common disease that involves leakage of urine during coughing, sneezing, or other increases in intraabdominal pressure, which is associated most often with pelvic organ prolapse（POP）. It is defined as "a complaint that the involuntary leakage of urine on effort or exertion, or on sneezing or coughing" and become a common problem in the female population, especially in ageing countries. It is estimated that SUI affects 40% of women, with> 300 000 anti-incontinence procedures performed annually in the USA, while 1/3 of them seek for another because of recurrence. A report revealed that 46.5% women of 18 or over suffer from urinary incontinence in Beijing, while 59.6% of which afflicted by SUI.Although the etiology of SUI is still poorly understood, in general, it is likely to be caused by a combination of anatomical support abnormalities and intrinsic urethral sphincter deficiency. The main risk factors include age, pregnancy, childbirth, obesity and so on. Moreover, high weight index, constipation, chronic coughing, smoking, family history and the level of estrogen may be partly responsible for SUI. The factors above lead to mechanical failure of urinary control, arousing a low pressure reservoir of the urethral sphincter and the involuntary leakage of urine. As well, SUI also can becaused by physical injuries.The fascial and muscular components within the pelvic floor create a dynamic support mechanism that facilitates storage and voiding of urine and faeces. It is reported that 73% old women have ineffective muscle contraction. Hale DS et al also found that some of smooth muscle from women with SUI appeared to be atrophy, apomorphosis or apoptosis. Boreham et al discovered that women with POP had collagen degradation and elastic proteins decrease. In addition, the risk factors, such as vaginal delivery and birth trauma may cause pelvic floor injuries. As a support of urethra and bladder, the laterourethral tissues connect to the endopelvic fascia of the anterior vaginal wall and is sensitive to mechanical force. As it is known to all, calpain does participate in the degradation of smooth muscle, collagen and other matrix of the connective tissue, so it may play a role in cause of SUI.Calpain is a calcium (Ca²⁺)-dependent cysteine protease. There are two classes of calpains: one （comprising calpains 1, 2, 5, 7, 10, 13, and 15） is ubiquitous in cytosol; the other （comprising calpains 3, 6, 8, 9, 11, and 12） occurs only or mainly in certain tissues. The two ubiquitous isoforms, micro-calpain （μ-calpain or calpain-1） and milli-calpain （m-calpain or calpain-2）, are different in their sensitivity to Ca²⁺. In vitro analysis has shown that the Ca²⁺ concentration required for optimal activity is 5-50μmol for calpain-1 and 0.2-1mmol for calpain-2. Each of them consists of two different polypeptide subunits. The larger subunit （80 KD） has catalytic activity, whereas the smaller, 30kD, subunit has a regulatory function. More than 50 endogenous and exogenous inhibitors of the calpains have been described. They include cellular and extracellular proteins and drugs such as iodoacetate, iodoacetamide, and N-ethylmaleimide, which are inhibitors of cysteine proteases. The specific endogenous inhibitor of calpain activity is calpastatin, which binds specifically to both isoforms of calpain in a substrate competitive manner. Theintracellular level of calpastatin correlates directly with calpain activation, and the affinity of calpastatin for the activated forms of the calpains is greater than its affinity for the proenzyme.Calpains cover a broad range of physiological functions including proteolysis of molecules involved in cytoskeletal organization, the cell cycle, signal transduction, apoptosis, and protein renewal during growth and tissue regeneration. While being pathologically activated, they would lead to diseases. A number of pathologic conditions have been associated with disturbances of the calpain system. They include type 2 diabetes, cataracts, Duchenne’s muscular dystrophy, Parkinson’s disease, Alzheimer’s disease, rheumatoid arthritis, ischemia, stroke and brain trauma, various platelet syndromes, hypertension, liver dysfunction, and some types of cancer. The application of calpain inhibitors may provide greater specificity and therapeutic potential.In this study, we investigated the expression of calpain-1, calpain-2 and their inhibitors, calpastatin in the human laterourethral tissues to show the potential link between calpain system and SUI, expecting to find a sensitive evaluation index or a potential treatment.Methods and ResultsMethods: The laterourethral tissues of 39 women with SUI and 31 women without SUI were collected to detect the expression of calpains and calpastatin using a semi-quantitative competitive RT-PCR and westblotting. The SUI group were diagnosised by SUI with no hormone treatment. The control group were selected from people with benign gynecological but not hormone releated diseases. Results: （1） There are calpain-1 mRNA and protein expresstions in both groups and the numbers are no differences between the two groups. （2） There are calpain-2mRNA and protein expresstions in both groups, and numbers of mRNA and protein expressions in the group of SUI are more than those in the control group （1.36±0.06 versus 1.16±0.04, p<0.05 and 0.75±0.07 versus 0.56±0.06, p <0.05）. （3） There are calpastatin mRNA and protein expresstions in both groups, the mRNA expressions in women with SUI are more than women without SUI （0.85±0.07 versus 0.62±0.06, p <0.05）, while the protein expressions are significantly less in them than in women without SUI. （0.72±0.06 versus 1.84±0.09,p <0.01）.Conclusions1. Both of calpain-2 mRNA and protein expression are higher in the people with SUI2.The expression of calpastatin mRNA is higher in the people with SUI than the control group, while the protein expression are lower than that in the control group.3. Over expression of calpain-2 and low expression of calpastatin may be play an important role in the cause of SUI, the application of calpastatin may be a new method to treat SUI.更多还原