【作者】 张晓丹；

【导师】 王鲁文；

【作者基本信息】 郑州大学 ， 妇产科学， 2011， 硕士

【摘要】 尿失禁是妇女的一个常见症状,它包括多种类型,如真性尿失禁、功能性尿失禁、压力性尿失禁、急迫性尿失禁等。在这些疾病中,以压力性尿失禁(stress urinary incontinence, SUI)最常见,其发病率约占尿失禁总发病率的50%-70%。目前,国内外对于SUI的定义如下：当增加腹压甚至在休息时,膀胱颈和尿道不能维持正常的压力,而有尿液不自主溢出的现象。SUI严重影响妇女的生活质量,随着世界人口老龄化程度的加重,SUI越来越多的受到广大学者的重视,为从根本上防范SUI的发生,学者对其病因进行了广泛的研究,但是目前尚未得到一致结论。盆底支持组织的衰弱与SUI的发生密切相关。弹性蛋白(Elastin)、胶原蛋白、层粘连蛋白等是盆底支持组织细胞外基质(extracellular martrix, ECM)的主要成分。有研究发现,胶原蛋白的含量、结构、功能、Ⅰ/Ⅲ型胶原相应比例异常与SUI的关系密切。另有研究证实,盆内筋膜代谢活跃,胶原的分解增加可能参与SUI形成。而弹性蛋白也是盆底支持组织中的一个主要成分,它能使所在组织具有伸缩性和可逆的变形能力,对于维持盆底组织功能的维持也有重大意义,目前Elastin在盆底器官脱垂(pelvic organ prolapse, POP)成因方面有所涉及,但是在SUI的发病原因方面国内外尚未有研究。基质金属蛋白酶(matrix metalloproteinase, MMPs)是调节ECM代谢的重要酶类,在盆底支持组织合成及分解代谢方面起着直接或间接的作用。MMPs是一组含Zn2+的蛋白酶家族,它能够降解ECM和基底膜,包括以下五大类：(1)胶原酶(MMP-1, MMP-8);(2)膜型蛋白酶(MT1-MMP、MT2-MMP等)；(3)明胶酶(MMP-2和MMP-9)；(4)基质溶解素(MMP-3, MMP-7, MMP-10);(5)弹性蛋白酶(MMP-12)。其中MMP-12的主要作用底物是弹性纤维、层粘连蛋白、纤粘连蛋白等。已有大量研究表明MMP-12在食管癌、上皮性卵巢癌、肺癌及结肠癌等疾病中均呈高表达,因人类女性阴道壁组织中含有弹性纤维,推测MMP-12与女性SUI的发病机制也有关联。基质金属蛋白酶抑制剂(tissue inhibitors of metalloproteinases, TIMPs)是一个多基因家族的编码蛋白对MMPs有负调节作用。TIMP-1是MMP-12的特异性抑制剂,在生理状态下,二者保持平衡,使体内Elastin的破坏与重建维持在正常水平。而当这种平衡状态被打破时,会引发体内一系列的病理情况。因此推测体内MMPs与TIMPs调节失衡也是SUI发病的原因之一。目的本课题取人阴道前壁组织为实验对象,用免疫组织化学方法检测各实验组对象阴道壁组织中MMP-12、TIMP-1、Elastin的含量,并对三指标之间的相关性进行分析,同时用RT-PCR法检测各组中Elastin mRNA的含量。了解MMP-12 TIMP-1、Elastin的表达与女性SUI之间的关系,以期今后能够从病因上为SUI的治疗提供依据。材料与方法1材料选取2009年8月到2010年2月在郑州大学第三附属医院妇科住院治疗的SUI患者(SUI组)30例,年龄在37-60岁之间,平均55.30±5.87岁；POP患者(POP组)30例,年龄在40-59岁之间,平均53.31±5.93岁；并选择非卵巢功能性肿瘤及宫颈上皮内瘤变患者30例作为对照组,在38-65岁之间,平均55.43±4.48岁。选取标准：SUI组：有尿失禁症状,根据妇科检查、各项辅助检查如尿动力学检查和尿垫试验等确诊为SUI,并排除不稳定膀胱、充盈性尿失禁及尿路感染等情况；POP组：患者自述无SUI症状,妇科检查有中度及以上阴道前壁膨出,辅助检查均证实无尿失禁；对照组：无尿失禁症状,妇科检查无盆底器官膨出,术后病理检查证实为非雌激素相关性疾病。取材部位：均选取阴道前壁上段12点处组织,大小约0.5cm×0.5cm×0.5cm。SUI组采用TVT-O、TVT-S手术或者阴道前壁修补术,POP组患者采用前盆底悬吊术、阴道前壁修补术,对照组采用子宫全切术(阴式或腹式)。经样本均衡性测定,三组病人在产次、年龄、体重指数等方面差异均无显著性(P>0.05)具有可比性。患者术前均签署知情同意书。2方法采用免疫组化S-P法,操作步骤遵循于试剂盒说明书,检测三组患者阴道壁组织中MMP-12、TIMP-1、Elastin的表达情况,并同时使用RT-PCR方法检测三组患者阴道壁组织中的Elastin RNA的含量,从分子水平研究Elastin的表达情况。3结果判定和图像分析3.1免疫组化法于显微镜下观察,阴道壁粘膜下结缔组织中各指标所表达部位呈现淡黄色至棕黄色颗粒沉着为阳性表达,MMP-12及TIMP-1主要定位在胞浆,Elastin主要定位于间质。全面观察每张切片中各指标的表达情况,从中随机选取5个有代表性的高倍视野(40×10倍),电子计算机图像分析仪计算分析各免疫组化染色阳性区的平均灰度值。染色强弱与平均灰度值之间成正比。3.2 RT-PCR法电泳后,照相记录,以内参基因β-actin为基准分析各条带的平均灰度值4统计学分析应用Excel 2003录入数据,各组数据的灰度值以均数±标准差(x±s)表示,用SPSS16.0软件包进行统计学分析。采用单因素方差分析比较三组间数据资料,用LSD-t检验进行组间两两比较,Pearson相关性分析比较各指标间的相关性。各种检验方法均以α=0.05为检验水准。结果1免疫组化法1.1各组患者阴道壁组织中MMP-12的表达MMP-12在3组阴道壁粘膜下结缔组织胞浆中都有表达,在SUI组与POP组的表达均比对照组强,差异有统计学意义(P<0.05)；而在SUI组与POP组之间的表达强度未见明显差别,差异无统计学意义(P>0.05)。1.2各组患者阴道壁组织中TIMP-1的表达TIMP-1在3组阴道壁粘膜下结缔组织胞浆中都有表达,在SUI组与POP组的表达均比对照组弱,差异有统计学意义(P<0.05)；而在SUI组与POP组之间的表达强度未见明显差别,差异无统计学意义(P>0.05)。1.3各组患者阴道壁组织中Elastin的表达Elastin在3组阴道壁粘膜下结缔组织间质中都有表达,在SUI组与POP组的表达均比对照组弱,差异有统计学意义(P<0.05)；而在SUI组与POP组之间的表达强度未见明显差别,差异无统计学意义(P>0.05)1.4 SUI组中MMP-12、TIMP-1、Elastin的相关性分析对SUI组阴道壁组织中三指标表达的平均灰度值进行相关性分析,结果显示：MMP-12与TIMP-1之间呈显著负相关(r=-0.999,P<0.05),MMP-12与Elastin之间呈显著负相关(r=-0.614,P<0.05),TIMP-1与Elastin的表达呈显著正相关(r=0.628,P<0.05)。2 RT-PCR法计算3组阴道壁粘膜中Elastin mRNA表达,在SUI组与POP组的表达均比对照组弱,差异有统计学意义(P<0.05)；而在SUI组与POP组之间的表达强度未见明显差别,差异无统计学意义(P>0.05)。结论MMP-12、TIMP-1可能通过对Elastin的调节参与SUI的发生更多还原

【Abstract】 Urine incontinence is a common symptom of female. There are many kinds of urine incontinence,such as true urine incontinence,functional urine incontinence, stress urine incontinence,urgent urine incontinence.Among the total,stress urine incontinence,which take an account of 50-70%,is the most common one.Stress urinary incontinence(SUI) is defined as the phenomenon when abdominal pressure increasing even when resting,bladder neck and urethral cann’t maintain certain pressure and urine overflow. It seriously affect women’s quality of life,with the aging population increase,SUI is more and more received the general scholars’ attention.Scholars have taken an extensive research fundamentally on its mechanisms to prevent SUI occurs,but haven’t obtained unanimous conclusion currently.SUI occurred closely related to the weakness of the pelvic support organization. Elastic protein(Elsatin), collagen, layer adhesion protein is main ingredient of extracellular matrix (ECM)’s. A study found that collagen content, structure, function, proportional anomalies ofⅠ/Ⅲcollagen type are closely related to SUI. Other studies have found that active fascia metabolic of pelvic support organizations and the increased decomposition of collagen might be involved in SUI formation. Elastin is also a main ingredient of pelvic support groups, it can make the organization has loft and reversible deformation ability. And it also has significant meaning to maintain strengthen the function of pelvic organizations. At present the Elastin has been reaserched in POP cause aspect somewhat, but in the SUI aspect has not been involved in at home and abroad.Matrix metalloproteinases(MMPs) is a group of important enzymes to adjust the metabolism of ECM and it plays a direct or indirect role in decomposed and anabolic aspects of pelvic support groups. MMPs is a group of protease family which contin Zn2+. It can degrade the extracellular matrix (ECM) and the basement membrane composition,its family includes the following five kinds of protease:(1) collagenase (MMP-1 and MMP-8), (2) membrane type protease (MT1-MMP, MT2-MMP etc.), (3) gelatinases (MMP-2 and MMP-9), (4) matrix dissolve hormone (MMP-3, MMP-7 and MMP-10), (5) elastase (MMP-12). Among them, the main substrate of MMP-12 is elastic fiber, layer adhesion protein and fibronectin etc. Now, a large studies had indicated that it is tending to high expression in patients with disease such as esophageal, epithelial oarian, lung and colon cancer. Because of vaginal wall tissue contains elastic fiber, presumably MMP-12 and female SUI pathogenesis also are linked.Matrix metalloproteinases inhibitors (inhibitors of tissue metalloproteinase, TIMPs) is a multigene family of coding protein.It have negative adjustment effect to MMPs. TIMP-1 is specific inhibitor to MMP-12.In physiological condition, they are both in a certain extent to keep balance which can make destruction and rebuilding of body elastin maintain normal state. When this balance is broken, a series of body pathological conditions can be caused. Therefore, imbalance between TIMPs and MMPs is also one of the reasons for the stress urine incontinence.ObjectiveThis topic take vaginal front wall organization of female as experimental subjects, using immunohistochemistry detecting content of MMP-12, TIMP-1, Elastin in each vaginal wall tissue, and analy the content of the correlation between the three indexes,And RT-PCR method to detect Elastin mRNA in each group.Understand relationship between MMP-12,TIMP-1, Elastin expressions and female stress urinary incontinence, in order to provides the basis to disease prevention and treatment in aspect of pathogeny in the future.Materials and Methods1 MaterialsAll patients are from The Third Affiliated Hospital of Zhengzhou University gynecological from 2009 August to 2010 February. With SUI patients 30 cases, Age between 37-60. POP patients 30 cases, Age between 40-59,and choose 30 patinents with cervical intraepithelial neoplasia or non-functional ovarian tumor as control group Age between 38-65.Selection standards:SUI groups:with SUI symptoms, be diagnosed as SUI according to gynaecological check-up, various auxiliary examination such as urinary dynamics inspection and urine mat test, and eliminate diagnosed unstable bladder, filling urinary incontinence, and urinary infection situation; etc. POP group:patients without stress incontinence symptoms, auxiliary examination both confirmed without incontinence, gynaecological check-up are moderate and above vaginal front wall surfaces.The control group:no incontinence symptoms, gynaecological check-up without pelvic organ prolapse. Postoperative histopathologic examination is proven to be an endometriosis etc estrogen related diseases.Based site:all take 12 points tissue of the anterior vaginal wall, 0.5cm×0.5cm×0.5cm. All selected patients didn’t take estrogen drugs for six months before operation. SUI group using TVT-O, TVT-S operation or vaginal front wall suture, POP group were treated by former strengthening POP or vaginal front wall suture group, uterine full cut method (Yin type or abdominal). The sample inner-layer determination, the three groups of patients in the age, production times, body mass index, etc non-significance difference (P>0.05), all have comparability. Preoperative patients were informed agreement signed.2 MethodBy immunohistochemical S-P method, in strict accordance with the kit manual method dyeing, detection three groups of vaginal wall organization MMP-12, TIMP-1, Elastin expression, and at the same time use RT-PCR method detection three groups of patients vaginal wall tissue of Elastin expression at the molecular level.3 Image and results analysis3.1 Immunohistochemical methodIn a microscope, in vaginal wall submucosal connective tissue, the place where the indexes expressed occurred pale to tan yellow particles as positive cells. MMP-12 and TIMP-1 are mainly engaged in cytoplasm, Elastin is mainly located in the stroma. Comprehensive observe each expression, randomly selected five representative meaning of magnification vision (10 by 40) from which slice, adopts electronic computer image analyzer analy and calculate the each positive immunohistochemical expression of average gray values, between dyeing strength and average gray values become direct ratio.3.2 RT-PCR methodAfter electrophoresis, photographic record, analy the average gary values with geneβ-actin prescriptions as a benchmark.4 Statistic analysisentry all data with Excel 2003, and establish database, each group of grey value are with mean±standard(x±s)deviation said. SPSS 16.0 packages were being applied to statistically analyzed. Using single factor analysis of variance comparison between three groups of data, with LSD-t-test for binary comparison between groups, Pearson correlation analysis comparing correlation between each index. Various inspection methods are toα=0.05 for inspection standard.Results1 Immunohistochemical method 1.1 MMP-12 expression in each vaginal wall organizationMMP-12 were expressed in three groups of vaginal wall submucosal connective tissue plasminogen. In SUI group and POP group expressions are stronger than the control group, and the intensity between the SUI group and POP group of expression has not seen the obvious difference. SUI group, POP ergometry respectively comparative differences are statistically significant (P<0.05). Be compared between SUI group and POP group, differences was not statistically significant (P>0.05).1.2 TIMP-1 expression in each vaginal wall organizationTIMP-1 were expressed in three groups of vaginal wall submucosal connective tissue plasminogen. In SUI group and POP group expressions are weaker than the control group, and the intensity between the SUI group and POP group of expression has not seen the obvious difference. SUI group, POP ergometry respectively comparative differences are statistically significant (P<0.05). Be compared between SUI group and POP group, differences was not statistically significant (P>0.05).1.3 Elastin expression in each vaginal wall organizationElastin were expressed in three groups of vaginal wall submucosal connective tissue plasminogen. In SUI group and POP group expressions are weaker than the control group, and the intensity between the SUI group and POP group of expression has not seen the obvious difference. SUI group, POP ergometry respectively comparative differences are statistically significant (P<0.05). Be compared between SUI group and POP group, differences was not statistically significant (P> 0.05).1.4 the correlation analysis of MMP-12,TIMP-1, Elastin in SUI groupSUI group of vaginal wall organization with MMP-12 TIMP-1 express mean grey value of the relevant analysis, the result shows:a significant negative correlation between (r=-0.999, P<0.05). SUI group of vaginal wall organization MMP-12 and Elastin express mean grey value of the relevant analysis, the result shows:a significant negative correlation between (r=-0.614, P<0.05). TIMP-1 and Elastin expression is a significant positive correlation (r=0.628, P<0.05). 2 RT-PCR methodCalculation Elastin mRNA expression of vaginal wall 3 groups, in SUI group and POP group expression is weaker than the control group. And between the SUI group and POP group has not seen the obvious difference. SUI group, the POP group compared with controls are statistically significant difference (P<0.05), SUI group and POP group between comparative differences was not statistically significant (P>0.05).ConclusionMMP-12、TIMP-1 may participate the occurence of SUI by regulate content of Elastin.更多还原