逼尿肌细胞中瞬时受体电位通道蛋白C6亚型的功能鉴定及其在膀胱过度活动症发病机制中的作用研究

Overactive bladder (OAB) syndrome is defined as urinary urgency, usually accompanied by frequency and nocturia, with or without urge urinary incontinence, in the absence of urinary tract infection or other obvious pathology. OAB seriously affects the patient's physical and mental health and quality of life, but the pathogenesis is little known. Muscarinic cholinergic receptor (MR) and calcium ions play very important roles in the detrusor contractility, while the mechanism of linking mechanism between two aspects in OAB patients is still not clear. In the previous study, we found that TRPC6 is over-expressed in the OAB detrusor. The current induced by MR-agonist 1-oleoyl-2-acetyl-sn-glycerol (OAG) was inhibited when TRPC6 protein was downregulated in bladder smooth muscle cells, and calcium influx was significantly reduced, which indicated that TRPC6 may be one of the important cation channel affecting the calcium influx of detrusor cells. Based on these findings, we hypothesized that TRPC6 overexpression induced by MR activation may regulate the Ca2 + influx and bladder smooth muscle cell contraction. The mechanism might contribute to the pathogenesis of OAB. In this study, we will use the patch-clamp and calcium imaging to detect the electric physiological properties of TRPC6 channel in detrusor, and purify the calcium influx factors by anion exchange high performance liquid chromatographic analysis technology. We will establish TRPC6 high expression and low expression cell lines by plasmid transfection and siRNA, and then observe the effect of TRPC6 on linking MR activation and calcium influx in bladder smooth muscle cells contraction. At the next step, we will detect the possible combination of MR and TRPC6 by co-immunoprecipitation. Additionally, we establish the OAB rat model to observe the relationship between TRPC6 as well as MR and the change of calcium influx in bladder smooth muscle contraction and explore the possibility of intervention and treatment of OAB in vivo by TRPC6 knockout rats. In this study, we verified the assumption from in vivo and in vitro experiments. We will study the biological characteristics of TRPC6 channel protein in bladder smooth muscle cells. The conclusion may be important to understand the receptor - ion channel contraction regulatory theory in detrusor cells under physiological conditions and clarify the pathogenesis of OAB. In addition, the results will provide new clues for the treatment of disease and drug development.

膀胱过度活动症（OAB）的发病机制仍不明确，逼尿肌细胞的胆碱能受体（MR）与钙离子通道的高表达是重要的病理改变，但二者之间的联系机制尚不清楚。在前期研究工作基础上，我们提出假设阳离子通道蛋白TRPC6，在MR活化后调控Ca2+内流及逼尿肌细胞收缩过程中起重要作用。本研究应用膜片钳及钙成像技术检测逼尿肌细胞中TRPC6通道的生理学特性；阴离子交换高效液相色谱分析技术提纯钙内流因子；质粒转染及siRNA技术，观察MR激活TRPC6对钙内流及逼尿肌细胞收缩的影响，免疫共沉淀检测二者的结合；构建OAB大鼠模型，同时利用TRPC6基因敲除大鼠模型，探索体内干预治疗OAB的可能。本研究从体内和体外两个水平验证假设，深入研究TRPC6通道蛋白在膀胱逼尿肌中的生物学特性，其结论对于理解生理条件下逼尿肌细胞的受体-离子通道调控理论，明确OAB发病原因，寻找相应的治疗措施有重要意义。

膀胱过度活动症（OAB）的发病机制仍不明确，逼尿肌细胞的胆碱能受体（MR）与钙离子通道的高表达是重要的病理改变，但二者之间的联系机制尚不清楚。在前期研究工作基础上，我们提出假设阳离子通道蛋白TRPC6，在MR活化后调控Ca2+内流及逼尿肌细胞收缩过程中起重要作用。本研究应用膜片钳及钙成像技术检测逼尿肌细胞中TRPC6通道的生理学特性；阴离子交换高效液相色谱分析技术提纯钙内流因子；质粒转染及siRNA技术，观察MR激活TRPC6对钙内流及逼尿肌细胞收缩的影响，免疫共沉淀检测二者的结合；构建OAB大鼠模型，同时利用TRPC6基因敲除大鼠模型，探索体内干预治疗OAB的可能。本研究从体内和体外两个水平验证假设，深入研究TRPC6通道蛋白在膀胱逼尿肌中的生物学特性，其结论对于理解生理条件下逼尿肌细胞的受体-离子通道调控理论，明确OAB发病原因，寻找相应的治疗措施有重要意义。