Restoration of Fecal Continence in Aging IAS

老年 IAS 患者大便失禁的恢复

基本信息

批准号：
7901968
负责人：
KHALIL N BITAR
金额：
$ 3.02万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-15 至 2010-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7901968
关键词：
3-Dimensional Acetylcholine Actins Adrenergic Agents Adult Affect Age Aging Anus Applications Grants Area Biomedical Engineering Canis familiaris Caveolae Caveolins Cell Aging Cells Circular layer of muscularis propria of anal canal Complementary DNA Complex Contractile Proteins Contracts Cyclic AMP Cytoskeletal Modeling Cytoskeleton Data Development Dose Elderly Enteric Nervous System Exhibits Fecal Incontinence Feces Functional disorder Gastrointestinal Contents Gastrointestinal Sphincter Generations HSPB1 gene Hormonal Human In Vitro Investigation Length Mechanics Mediator of activation protein Membrane Microdomains Modeling Molecular Muscle Muscle Cells Muscle Contraction Muscle function Nerve Phosphorylation Physiological Play Property Protein Isoforms Rattus Rectum Regulation Relaxation Rest Role Signal Transduction Skeletal Muscle Smooth Muscle Smooth Muscle Myocytes Sphincter Time Tissue Engineering Work adrenergic age effect age related aged caveolin 1 cell age cholinergic in vitro Model in vivo novel overexpression pressure response restoration skeletal

项目摘要

DESCRIPTION (provided by applicant): Little is known about the mechanisms or pathophysiology responsible for age-related decline of internal anal sphincter (IAS) function. Decreased mechanical efficiency of smooth muscle of the IAS results in decreased closure pressure of the sphincter, thus greatly contributing to fecal incontinence which is disproportionately prevalent in the elderly. Recent advances in tissue engineering provide us with an excellent in vitro model mimicking in vivo function to study the effects of aging on the molecular mechanisms of the IAS smooth muscle. We have for the first time, bioengineered three- dimensional (3-D) rings from isolated smooth muscle cells from human IAS. These rings developed tone, responded to acetylcholine in a dose-dependent manner and relaxed upon the exogenous addition of the relaxant mediator 8-Br-cAMP. Preliminary results from Human IAS and Human circular colonic smooth muscle cells (CSMC) indicate: 1) Sequestration of RhoA, phospho-PKC1 (S657) and HSP27 only in the caveolin-rich lipid raft microdomains of IAS cells at rest and not CSMC; 2) a greater expression of HSP27, RhoA, PKC1 and phospho CPI-17 in Human IAS cells vs. CSMC. Rings from old Rat IAS cells showed decreased contractile response (maximal force generation 5N and time-to-peak response) when compared to IAS rings from adult rats that correlated with decreased HSP27 phosphorylation. Reduced phosphorylation of HSP27 affects actin cytoskeleton stability leading to disturbed caveolae formation. Overexpression of phosphomimic-HSP27 in old IAS smooth muscle cells exhibited increased association of PKC1 and HSP27 with caveolin-1, and also reinstated the magnitude of force generated and the time-to-peak contraction in IAS rings bioengineered from these cells. The specific aims of this grant proposal are: 1) Develop a 3-D physiological model of the IAS bioengineered in vitro from cells isolated from the IAS of human and of adult and aged rats, and examine the effect of aging on the molecular mechanisms of tonic IAS smooth muscle contraction 2) Study the role of phosphorylated-HSP27 in age-related decline of IAS smooth muscle function, and 3) Examine the reinstatement of physiological contractile function in 3-D IAS rings bioengineered from IAS smooth muscle cells transfected with phosphomimic-HSP27 cDNA.

描述（由申请人提供）：对于与年龄相关的肛门内括约肌（IAS）功能下降的机制或病理生理学知之甚少。 IAS平滑肌机械效率降低导致括约肌闭合压力降低，从而极大地导致大便失禁，这在老年人中尤为普遍。组织工程的最新进展为我们提供了一个模拟体内功能的优秀体外模型，以研究衰老对 IAS 平滑肌分子机制的影响。我们首次从人类 IAS 中分离出的平滑肌细胞中获得了生物工程三维 (3-D) 环。这些环产生音调，以剂量依赖性方式响应乙酰胆碱，并在外源添加松弛介质 8-Br-cAMP 后松弛。人 IAS 和人环状结肠平滑肌细胞 (CSMC) 的初步结果表明：1) RhoA、磷酸化 PKC1 (S657) 和 HSP27 仅隔离在静息状态 IAS 细胞富含小窝蛋白的脂筏微域中，而 CSMC 中不存在； 2) 与 CSMC 相比，人 IAS 细胞中 HSP27、RhoA、PKC1 和磷酸化 CPI-17 的表达更高。与来自成年大鼠的 IAS 环相比，来自老年大鼠 IAS 细胞的环显示出收缩反应降低（最大力产生 5N 和达到峰值反应的时间），这与 HSP27 磷酸化的降低相关。 HSP27 磷酸化的减少会影响肌动蛋白细胞骨架的稳定性，从而导致小凹形成受到干扰。在旧的 IAS 平滑肌细胞中过度表达拟磷酸化-HSP27 表现出 PKC1 和 HSP27 与 Caveolin-1 的关联增加，并且还恢复了由这些细胞生物工程改造的 IAS 环产生的力的大小和达到峰值的收缩时间。该拨款提案的具体目标是：1）利用从人类以及成年和老年大鼠的 IAS 中分离的细胞，开发体外生物工程的 IAS 3D 生理模型，并检查衰老对其分子机制的影响。强直 IAS 平滑肌收缩 2) 研究磷酸化 HSP27 在 IAS 平滑肌功能与年龄相关的衰退中的作用，以及 3) 检查生物工程 3-D IAS 环中生理收缩功能的恢复来自用拟磷-HSP27 cDNA转染的IAS平滑肌细胞。