成纤维细胞中雌激素调控的Aquaporin 2与女性压力性尿失禁发病机制的研究

With the advent of aging society and the pursuit of the high quality of life, female stress urinary incontinence (SUI), as a common chronic disease of women, is attracting more and more attention. Although SUI has been shown to increase with age and is often associated with menopause, the delicate mechanism of SUI induced by estrogen deficiency following menopause is still unclear. Extensive research has demonstrated abnormal extracellular matrix (ECM) remodeling of female periurethral connective tissues, which could be induced by estrogen deficiency following menopause, may play a critical role of the pathogenesis of SUI.And the ECM remodeling is mainly induced by the activities of fibroblast (migration, proliferation, synthesis and secretion of collagen, etc.). ..It is now evident that aquaporins (AQPs) are closely related to the process of cell migration and wound healing, and our previous studies have revealed that estrogen could directly regulate the AQP2 expression. We also found the expression of AQP2 in fibroblasts of female periurethral connective tissues from postmenopausal patients significantly decreased compared with that in premenopausal patients. However, the regulation mechanism is unclear. We believe that AQP2 may be involved in the pathogenesis of SUI induced by estrogen deficiency.This project aims to verify these relationships through clinical experiments and in vitro cellular experiments...First,to confirm the correlation with low AQP2 expression and SUI in postmenopause.We will analyze the mRNA and protein expression of AQP2 in fibroblast of female periurethral connective tissues from premenopausal and postmenopausal patients. Second, to investigate the effect of estrogen on migration, proliferation and ECM secretion of fibroblast，and the role of AQP2.We will inhibit the expression of AQP2 in the fibroblast cell line using siRNA techniques. Migration capacity will be determined by scratch test and cell pseudopodia observed. Proliferation capacity will be determined by flow cytometry assay and MTT assay. ECM secretion will be determined by procollagen expression (analyzed by real-time PCR and ELISA) and secretory vesicles observed. Third, to investigate the mechanism of AQP2 in fibroblast activities. We will observe the changes of migration, proliferation and ECM secretion of fibroblast using over-expression or inhibition of AQP2 in fibroblast cell lines. Changes of some molecules which are important in cell migration (eg.F-actin, Annexin2) ,exocytosis (eg.VAMP, syntaxin, SNAP-25) and cell proliferation &ECM metabolism (eg.TGF-β1，IL-1? ) will be evaluated using real-time PCR, Western blot, ELISA and immunohistochemistry, respectively. ..These experiments will enable insights into the molecular mechanisms of SUI. In the future, these results may provide novel AQP2-based strategies for early management of SUI.

压力性尿失禁（SUI）是严重影响中老年妇女生活质量的常见病，随着经济发展和人口老龄化，该病已引起整个医学界的重视。绝经是其发病的高危因素之一，但确切机制不清。多数学者认为，绝经后雌激素低下引起的尿道支撑组织细胞外基质（ECM）重构障碍可导致SUI发生，而ECM重构由成纤维细胞迁移、功能活化来主导。水通道蛋白（AQPs）与细胞迁移、创伤修复密切相关。在前期研究中，我们课题组已证实雌激素直接调控水通道蛋白2（AQP2）的表达，并首次发现女性尿道支撑组织成纤维细胞中存在AQP2，其表达下降与雌激素低下和SUI发病密切相关，但其调控机制不明。本项目拟通过临床实验和体外细胞实验，以研究成纤维细胞功能为切入点，采用AQP2过表达和干扰技术，阐明尿道支撑组织成纤维细胞AQP2表达对细胞迁移、增殖和ECM合成分泌功能的调控及机制。本课题将有望从新的视觉阐述SUI发病机制，并为SUI早期防治提供科学依据。

女性压力性尿失禁（SUI）是严重影响中老年妇女生活质量的常见病，绝经是其发病的高危因素之一，但确切机制不清。多数学者认为，绝经后雌激素低下引起的尿道支撑组织细胞外基质（ECM）重构障碍可导致SUI发生，而ECM重构由成纤维细胞迁移、功能活化来主导。水通道蛋白（AQPs）与细胞迁移、创伤修复密切相关。在前期研究中，我们课题组已证实雌激素直接调控水通道蛋白2（AQP2）的表达，并首次通过Western blot法发现绝经后SUI患者尿道支撑组织AQP2蛋白表达水平较非SUI者下降，通过免疫组化法发现绝经后SUI患者中该组织成纤维细胞上AQP2表达水平较非SUI者下降，但具体机制不清。本项目基于此研究结果，对女性尿道支撑组织成纤维细胞进行原代培养，通过western blot法及免疫荧光法明确了AQP2在该成纤维细胞中的表达及定位，利用AQP2过表达和干扰技术，发现AQP2过表达可显著增加Ⅰ型和Ⅲ型胶原的表达，但对细胞迁徙和细胞增殖无明显作用，且AQP2对女性尿道支撑组织成纤维细胞的胶原代谢作用不受外源性雌激素干扰的影响；进而探讨AQP2对胶原代谢的调节机制，发现AQP2促进尿道支撑组织成纤维细胞SNAREs核心复合体的表达，抑制TGF-ß1和 IL-1ß蛋白分泌。通过本项目研究，我们明确了AQP2在女性尿道支撑组织及其成纤维细胞中的强表达并发现其表达水平与绝经后SUI发病可能相关，阐明了AQP2在以I型、III型胶原为主的细胞外基质参与的女性尿道支撑组织损伤、修复过程中具有调节胶原代谢的作用，但该作用可能是通过SNAREs核心复合体及TGF-ß1和 IL-1ß信号通路而与雌激素-AQP2分子通路无明确相关性。本项目研究结果提示，继续探讨AQP2对女性尿道支撑组织成纤维细胞胶原代谢的调控作用及其具体分子学机制是研究女性SUI的发病机制及寻求该病早期防治措施的一条可行的思路。 在本项目执行过程中，已培养了硕士研究生2名，发表了SCI论文1篇，待发表SCI论文1篇，组织了1次妇科泌尿学术会议。