MECHANICAL REGULATION OF THE IGF SYSTEM IN THE BLADDER

膀胱出口梗阻是神经源性、前列腺、先天性和/或尿道狭窄疾病的常见并发症。它的特点是膀胱平滑肌质量增加，膀胱容量和大小和尿潴留。细胞产生的力量减少，膀胱功能往往不可逆转地受损，尽管缓解梗阻。机械应变是触发膀胱平滑肌细胞（SMC）生长和合成表型变化的最可能的刺激类型。缺乏足够水平的机械运动或大量工作，或膀胱壁正常拉伸模式的破坏，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)，以及可能参与调节其生物活性的蛋白质，包括受体和结合蛋白。本研究的目的是确定机械刺激在体外和体内影响IGF系统成分表达的程度，并提供有关膀胱中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，将在培养的膀胱SMCs中检测菌株水平对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仅仅存在一个阈值。这些变化对细胞生长和基质蛋白合成的功能意义也将进一步研究。第二个目标是研究igf - 1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'侧翼区域的功能作图来研究，重点是鉴定在菌株反应中至关重要的区域。第三个目标是在胎儿动物模型中检查IGF系统成分的表达和分泌特征，在胎儿动物模型中，正常的膀胱壁力学因部分出口阻塞和膀胱阻塞逆转而改变。基因表达的变化将通过Northern blots、核糖核酸酶保护试验、western配体blotting和放射免疫试验在mRNA和蛋白质水平上进行评估。DNA转染实验和DNA-蛋白质相互作用分析将用于定义启动子区域的拉伸调控序列和反式作用因子。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.