TGF-β1、Smad3和CTGF在女性压力性尿失禁患者阴道壁组织中的表达及意义

发布时间：2018-05-14 20:39

本文选题：压力性 + 尿失禁　；参考：《郑州大学》2014年硕士论文

【摘要】：压力性尿失禁(Stress Urinary Incontinence，SUI)被认为是尿道对控尿起“开关-阀门”失控引起的功能障碍性疾病，多数患者由于分娩、创伤等引起肛提肌损伤、盆底筋膜和韧带松弛、泌尿道解剖位置改变所致。SUI是一种多因素参与的复杂性疾病，往往伴随女性盆腔器官脱垂（pelvic organ prolapse，POP）。其特点是平时无漏尿，如咳嗽、大笑、重体力劳动等腹压增加时，尿液不自主地自尿道流出，且症状严重程度随年龄的增长而加重，在美国60岁及以上38%妇女及17%男性均患有尿失禁。因此，国际控尿协会将SUI定义为腹压突然增加时导致尿液失控不自主流出，其病因非逼尿肌收缩压引起而是盆底解剖结构出现异常或缺损致压力传导不均。有人将该病形象的称为“社交癌”。虽然压力性尿失禁不像心血管、肿瘤等疾病严重危及患者的生命健康，却也引起了社会、经济和卫生等不同程度的影响；其严重程度越来越受到广大妇女的关注。随着“吊床假说”和“盆底整体理论”的提出，人们逐渐意识到仅单纯检测尿道压、尿道长度及膀胱尿道角等解剖改变的传统方法已经无法全面合理解释SUI的发病机制，这就促使研究人员从分子生物学水平深入研究盆底支持组织的解剖结构及功能。众多研究表明，女性SUI患者盆底支持结构中胶原及弹性蛋白含量显著减少，引起支持组织弹性减弱，出现盆底松弛。而转化生长因子-β1（TGF-β1）作为最重要的细胞因子，既与胶原和弹性蛋白的合成及降解直接相关，又可改变基质降解酶及其抑制因子的量及活性，在分娩、手术等造成盆底支持组织损伤修复过程中发挥关键性作用。结缔组织生长因子（CTGF）可刺激成纤维细胞增殖和分泌胶原，在纤维化性疾病、创伤后瘢痕形成等方面有较多的研究，然而在盆底障碍性疾病的研究国内外少有报道。而Smad3蛋白是细胞内TGF-β信号转导分子，对细胞功能起着双向调控作用，参与控制创面组织的愈合。目的本研究通过采用免疫组化和聚合酶-逆转录（RT-PCR）方法检测TGF-β1、CTGF及Smad3三者在女性SUI患者阴道壁组织中的表达，分析三者之间的相关性，探讨他们在SUI发生机制中的作用，期望为该病的预防、治疗提供理论依据。材料和方法 1.材料选取2012年6月-2013年4月在本院妇产科收治的的60例中、重度压力性尿失禁患者为实验组，包括25例单纯性SUI，35例SUI合并POP，手术方式为经阴道无张力尿道中段悬吊术（trans-obturator vaginal tape，TVT-O）、阴道前后壁修补术，阴道前壁悬吊术。同期选取20例宫颈上皮内瘤变患者均不伴SUI为对照组，手术方式为腹式或阴式子宫切除术。3组在年龄、产次及体重指数等方面差异无统计学意义（P0.05），具有较好的可比性。所有病例均无类风湿性关节炎、甲状旁腺功能亢进、肺气肿和肝纤维化等影响胶原代谢的疾病，术前3个月内未曾使用过性激素类药物，未患功能性卵巢肿瘤，无泌尿生殖道感染，无既往盆底手术史。SUI诊断依据为患者的病史、妇科检查、压力试验、指压试验、尿垫实验及尿动力学检查结果。SUI合并POP诊断依据是除具有压力性尿失禁的诊断标准外，妇科检查均发现合并有POP-Q分类法Ⅱ度及Ⅱ度以上的阴道前壁膨出。实验组于阴道前壁12点或膀胱宫颈韧带周围取两块阴道前壁组织各约0.8cm×0.5cm×0.2cm大小。对照组取自子宫切除后阴道残端阴道壁组织。 2.实验方法采用免疫组化（SP）法检测SUI组、SUI+POP组和对照组患者阴道前壁组织中TGF-β1、CTGF和Smad3蛋白的表达情况。采用逆转录-聚合酶链式反应（RT-PCR)技术检测三组TGF-β1、CTGF和Smad3mRNA水平。 3.统计学分析采用SPSS17.0统计软件对数据处理。多组定性资料的比较采用独立的多组二分类，两两比较采用四格表卡方检验，检验水准取α/3=0.0167；多组定量资料的比较采用单因素方差分析，组间比较采用LSD-t法。两连续变量相关分析采用Pearson积矩相关。检验水准α=0.05。结果 1．免疫组化染色结果 SUI组、SUI+POP组和对照组3组患者阴道壁组织中均可见TGF-β1蛋白、CTGF蛋白和Smad3蛋白的表达。TGF-β1和CTGF蛋白主要表达于胞浆，呈现淡黄色至棕黄褐色的细胞为阳性细胞；Smad3蛋白主要表达于细胞核，呈现淡黄色至棕黄褐色颗粒的细胞为阳性细胞。 1) TGF-β1蛋白在3组阴道壁中阳性表达率分别为28%、17.14%、95%，3组之间差异有显著性（χ2=34.327，P0.01）；SUI组和SUI+POP组比较，差异无显著性（P0.05）。 2) CTGF蛋白在3组阴道壁中阳性表达率分别为16%、20%、95%，3组之间差异有显著性（χ2=37.717，P0.01）；SUI组和SUI+POP组比较，差异无显著性（P0.05）。 3) Smad3蛋白在3组阴道壁中阳性表达率分别为24%、14.29%、90%，3组之间差异有显著性（χ2=33.933，P0.01）；SUI组和SUI+POP组比较，差异无显著性（P0.05）。 2. RT-PCR结果半定量测定结果显示：TGF-β1mRNA、CTGFmRNA及Smad3mRNA在3组中均为阳性表达。 1) TGF-β1mRNA在SUI组、SUI+POP组和对照组阴道壁组织中表达量分别为（0.294±0.013）、（0.293±0.019）和（0.610±0.052），SUI组、SUI+POP组与对照组比较差异均有显著性（P0.01）；而SUI组与SUI+POP组比较差异无显著性（P0.05）。 2) CTGF mRNA在SUI组、SUI+POP组和对照组阴道壁组织中表达量分别为（0.085±0.012）、（0.092±0.011）和（0.272±0.029），SUI组、SUI+POP组与对照组比较差异均有显著性（P0.01）；而SUI组与SUI+POP组比较差异无显著性（P0.05）。 3) Smad3mRNA在SUI组、SUI+POP组和对照组阴道壁组织中表达量分别为（0.091±0.011）、（0.088±0.012）和（0.272±0.029），SUI组、SUI+POP组与对照组比较差异均有显著性（P0.01）；而SUI组与SUI+POP组比较差异无显著性（P0.05）。 3. SUI患者组织中TGF-β1和CTGF的关联性分析相关分析显示：SUI组阴道壁组织中TGF-β1与CTGF呈正相关（r=0.959，P0.05），TGF-β1与Smad3的表达呈正相关（r=0.944，P0.05），CTGF与Smad3的表达呈正相关（r=0.965，P0.05）。结论 TGF-β1、CTGF及Smad3三者表达均降低，提示其可能参与了压力性尿失禁的发生。
[Abstract]:Stress urinary incontinence (Stress Urinary Incontinence, SUI) is considered to be a dysfunctional disease caused by the urethral "switch valve" out of control of the urinary tract. Most patients are caused by labor, trauma, and other injuries of the muscle of the anus, the pelvic floor fascia and ligamentum laxity, and the change of the urinary anatomic position caused by.SUI is a complex disease involved in multiple factors. The disease is often accompanied by pelvic organ prolapse (POP). It is characterized by urinary incontinence when no leakage of urine, such as coughing, laughter, and heavy manual labor, increases with age. Therefore, 38% women and 17% men in the United States have urinary incontinence. The International Association for control of the urine (SUI) defines an involuntary outflow of runaway urine resulting from a sudden increase in abdominal pressure. The cause of the cause is not detrusor contraction pressure, but an abnormal or defective stress conduction in the pelvic anatomy. Some people call the image "social cancer". Although stress urinary incontinence is not as serious as cardiovascular and tumor diseases Endanger the patient's life and health, but it also causes social, economic and health effects, and its severity is becoming more and more popular with women. With the "hammock hypothesis" and "pelvic floor holistic theory", people are gradually aware of the anatomic changes of urethral pressure, urethral length and bladder urethra angle. The traditional methods have been unable to explain the pathogenesis of SUI in a comprehensive and rational way. This has prompted researchers to study the anatomical structure and function of pelvic floor support from the molecular biology level. Many studies have shown that the content of collagen and elastin in the pelvic floor support structure of female SUI patients is significantly reduced, and the elasticity of support tissue is weakened and the basin is present. The growth factor - beta 1 (TGF- beta 1), as the most important cytokine, is not only directly related to the synthesis and degradation of collagen and elastin, but also changes the amount and activity of the matrix degrading enzyme and its inhibitory factors. It plays a key role in the repair of pelvic floor support injury in childbirth and surgery. Factor (CTGF) stimulates the proliferation and secretion of collagen in fibroblasts. There are many studies in the aspects of fibrotic diseases and posttraumatic scar formation. However, few reports have been made in the study of pelvic floor disorders at home and abroad. The Smad3 protein is a intracellular TGF- beta signal transduction molecule, which plays a bidirectional regulatory role in cell function and participates in the control of the wound. The healing of the tissue.
objective
In this study, the expression of TGF- beta 1, CTGF and Smad3 in the vaginal wall tissue of female SUI patients was detected by immunohistochemistry and polymerase chain reverse transcription (RT-PCR) method, and the correlation between the three groups was analyzed and their role in the pathogenesis of SUI was discussed. The theoretical basis was provided for the prevention and treatment of the disease.
Materials and methods
1. material
Among the 60 cases of severe stress urinary incontinence admitted in June 2012 -2013 and April in our department of Obstetrics and Gynecology, severe stress incontinence was included in the experimental group, including 25 cases of simple SUI, 35 cases of SUI combined with POP, and the operation method was transvaginal tension-free urethral suspension (trans-obturator vaginal tape, TVT-O), vaginal wall repair and anterior vaginal wall suspension. 20 cases of cervical intraepithelial neoplasia were selected without SUI as the control group. The surgical methods were abdominal or vaginal hysterectomy in group.3 with no statistically significant difference in age, birth order and body mass index (P0.05), and had better comparability. All cases had no rheumatoid arthrosis, hyperparathyroidism, emphysema and liver fibrosis. No sex hormone drugs have been used in 3 months before operation, no functional ovarian tumors, no urogenital tract infection, no history of previous pelvic floor surgery.SUI diagnosis is based on patients' history, gynecologic examination, stress test, pressure test, urinary cushion test and urodynamic examination results of.SUI combined with POP diagnosis basis Except for the diagnostic criteria for stress urinary incontinence, the gynecologic examination found the anterior vaginal wall bulge with POP-Q classification II and more than 2 degrees. The experimental group took about 0.8cm x 0.5cm x 0.2cm in the anterior wall of the vagina at 12 points of the anterior vaginal wall or around the cervix ligament of the bladder. The vaginal wall of the vaginal stump after hysterectomy was taken from the group. Organization.
2. experimental method
The expression of TGF- beta 1, CTGF and Smad3 in the anterior vaginal wall of group SUI, SUI+POP group and control group was detected by immunohistochemistry (SP). The reverse transcription polymerase chain reaction was used.
(RT-PCR) techniques were used to detect the levels of TGF- beta 1, CTGF and Smad3mRNA in the three groups.
3. statistical analysis
SPSS17.0 statistical software was used for data processing. Multiple groups of qualitative data were compared by independent multiple groups of two classifications, 22 compared with four lattice chi square tests and test level for alpha /3=0.0167; multiple quantitative data were compared by single factor analysis of variance and LSD-t method was used for comparison between groups. Two continuous variable correlation analysis adopted Pearson moment analysis. Correlation. Test level alpha =0.05.
Result
1. immunohistochemical staining results
TGF- beta 1 protein was found in the vaginal wall of group SUI, group SUI+POP and control group. The expression of.TGF- beta 1 and CTGF protein expressed mainly in the cytoplasm, and the positive cells were yellowish to brown brown cells, and Smad3 protein was mainly expressed in the nucleus, and the cells were yellowish to brown brown granules. Positive cells.
1) the positive expression rate of TGF- beta 1 protein in the 3 groups of vaginal walls were 28%, 17.14%, 95%, respectively. The difference between the 3 groups was significant (x 2=34.327, P0.01), and there was no significant difference between the SUI and SUI+POP groups (P0.05).
2) the positive expression rate of CTGF protein in the 3 groups of vaginal walls was 16%, 20%, 95%, and the difference between the 3 groups was significant (x 2=37.717, P0.01), and there was no significant difference between the SUI group and the SUI+POP group (P0.05).
3) the positive expression rate of Smad3 protein in the 3 groups of vaginal walls was 24%, 14.29%, 90%, and the difference between the 3 groups was significant (x 2=33.933, P0.01), and there was no significant difference between the SUI group and the SUI+POP group (P0.05).
2. RT-PCR results semi quantitative analysis showed that TGF- beta 1mRNA, CTGFmRNA and Smad3mRNA were all positive in the 3 groups.
1) the expression of TGF- beta 1mRNA in SUI group, SUI+POP group and control group was (0.294 + 0.013), (0.293 + 0.019) and (0.610 + 0.052), SUI group and SUI+POP group were significantly different from those of control group (P0.01), but there was no significant difference between group SUI and SUI+POP group (P0.05).
2) the expression of CTGF mRNA in SUI group, SUI+POP group and control group was (0.085 + 0.012), (0.092 + 0.011) and (0.272 + 0.029), SUI group and SUI+POP group were significantly different from those of control group (P0.01), but there was no significant difference between group SUI and SUI+POP group (P0.05).
3) the expression of Smad3mRNA in SUI group, SUI+POP group and control group was (0.091 + 0.011), (0.088 + 0.012) and (0.272 + 0.029), SUI group, SUI+POP group was significantly different from that of control group (P0.01), but there was no significant difference between group SUI and SUI+POP group (P0.05).
Correlation analysis between TGF- beta 1 and CTGF in 3. SUI patients
The correlation analysis showed that the TGF- beta 1 in the vaginal wall of SUI group was positively correlated with CTGF (r=0.959, P0.05), and TGF- beta 1 was positively correlated with the expression of Smad3 (r=0.944, P0.05), and there was a positive correlation between the expression of CTGF and Smad3 (r=0.965,).
conclusion
The expression of TGF- beta 1, CTGF and Smad3 decreased, suggesting that it may be involved in the occurrence of stress urinary incontinence (three).

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R711.59

【参考文献】