MECHANICAL REGULATION OF THE IGF SYSTEM IN THE BLADDER

膀胱出口梗阻是神经源性、前列腺源性、先天性和/或尿路狭窄疾病引起的一种常见并发症。它的特点是膀胱平滑肌质量增加，膀胱容量和大小增加，尿潴留。尽管梗阻解除了，但细胞力的产生减少了，膀胱功能经常受到不可逆转的损害。机械应变是最有可能引起膀胱内平滑肌细胞(SMC)生长和合成表型改变的刺激类型。缺乏足够水平的机械运动或容量功，或者膀胱壁内正常的拉伸模式被破坏，都会改变关键生长因子基因的表达，如胰岛素样生长因子-I(IGF-I)，并可能改变参与调节其生物活性的蛋白质，包括受体和结合蛋白。本研究的目的是明确机械刺激在多大程度上影响体外和体内IGF系统成分的表达，并提供有关膀胱这些变化的分子机制的新信息。这些目标将通过解决三个具体目标来实现。在第一个目标中，将检测不同应变水平对培养的膀胱SMC中IGF-I、IGF-I受体和IGF-I结合蛋白(IGFBP)基因表达的影响，以确定这些基因是否只存在一个阈值，超过这个阈值这些基因就会做出反应。这些变化对细胞生长和基质蛋白合成的功能意义也将被研究。第二个目标将研究IGF-I基因在转录或转录后水平对机械输入的潜在调节。转录调控将通过5‘侧翼区的功能图谱进行研究，重点是识别在应变反应中至关重要的区域。第三个目的是在一个胚胎动物模型中检测IGF系统成分的表达和分泌情况，在该动物模型中，部分出口梗阻和膀胱梗阻逆转改变了正常膀胱壁的力学结构。基因表达的变化将通过Northern印迹、核糖核酸酶保护分析、Western配体印迹和放射免疫分析在mRNA和蛋白质水平上进行评估。基因转染实验和DNA-蛋白质相互作用分析将用于确定启动子区域的拉伸调节序列(S)和反式作用因子(S)。免疫组织化学分析将确定这些蛋白质的组织和细胞定位。

Bladder outlet obstruction is a common complication that occurs as a result of neurogenic, prostatic, congenital and/or urethral stricture disease. It is characterized by increased bladder smooth muscle mass, bladder capacity and size and urinary retention. The cellular force generation decreases and bladder function is often irreversibly impaired despite the relief of the obstruction. Mechanical strain is the most likely type of stimulus that triggers changes in smooth muscle cell (SMC) growth and synthetic phenotype in the bladder. A lack of either an adequate level of mechanical exercise or volume work, or a disruption of the normal pattern of stretch within the bladder wall, alters the expression of key growth factor genes like the insulin-like growth factor-I (IGF-I) and potentially the protein involved in the regulation of its bioactivity including receptor and binding proteins. The objectives of this study are to define the extent to which a mechanical stimulus affects the expression of the IGF system components in vitro and in vivo and to provide new information regarding the molecular mechanisms responsible for these changes in the bladder. These goals will be achieved by addressing three specific aims. In the first aim, the effect of strain levels on IGF-I, IGF-I receptor and IGF-I binding protein (IGFBP) gene expression will be examined in cultured bladder SMCs to determine whether there is merely a threshold above which these genes respond. The functional significance of these changes on cell growth and matrix protein synthesis will also be studied. The second aim will examine the potential regulation of IGF-I gene at transcriptional or post-transcriptional levels in response to a mechanical input. Transcriptional regulation will be studied by functional mapping of the 5' flanking region with emphasis on the identification of regions that are crucial in the strain- response. The third aim will examine the expression and secretory profile of the IGF system components in a fetal animal model in which normal bladder wall mechanics were altered by partial outlet obstruction and after bladder obstruction reversal. Changes in gene expression will be evaluated at the mRNA and protein levels using Northern blots, ribonuclease protection assays, western ligand blotting and radioimmunoassays. DNA transfection experiments and DNA-protein interaction analyses will be used to define stretch-regulatory sequence(s) and trans- acting factor(s) in the promoter region. Immunohistochemical analysis will determine the tissue and cellular localization of these proteins.