Restoration of Fecal Continence in Aging IAS

老年 IAS 患者大便失禁的恢复

• 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
• 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

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）：对负责年龄相关的肛门括约肌（IAS）功能下降的机制或病理生理学知之甚少。 IAS平滑肌的机械效率降低导致括约肌的闭合压力降低，因此极大地导致了粪便尿失禁，这在老年人中非常普遍。组织工程的最新进展为我们提供了模仿体内功能的出色体外模型，以研究衰老对IAS平滑肌分子机制的影响。我们首次有来自人类IAS分离的平滑肌细胞的生物工程三维（3-D）环。这些环形成了音调，以剂量依赖性的方式对乙酰胆碱做出反应，并在外源添加弛豫的介体8-BR训练室后放松。人类IAS和人循环结肠平滑肌细胞（CSMC）的初步结果表明：1）仅在REST和CSMC的IAS细胞的富含小窝脂质细胞中的RhoA，RhoA，Phosho-PKC1（S657）和HSP27的隔离; 2）在人IAS细胞与CSMC中，HSP27，RhoA，PKC1和Phosho CPI-17的更大表达。与与HSP27磷酸化降低相关的成年大鼠的IAS环相比，来自旧大鼠IAS细胞的环显示收缩反应降低（最大力量生成5N和峰值响应）。 HSP27的磷酸化降低会影响肌动蛋白细胞骨架稳定性，导致小窝形成受到干扰。旧IAS平滑肌细胞中磷酸HSP27的过表达表现出PKC1和HSP27与Caveolin-1的缔合增加，并且还恢复了产生的力量和IAS峰值收缩的IAS响应中的峰值收缩，从这些细胞中生物加工。该赠款提案的具体目的是：1）开发一个从人类，成年大鼠和成年大鼠IAS分离的细胞中生物工程的IAS生物工程的3-D生理模型，并检查衰老对滋补肌肉平滑肌肉收缩的分子机制的影响2）研究在磷酸化的HSP27中的磷酸化和3次的作用。 3-D IAS环的生理收缩功能是由IAS平滑肌细胞生物工程的，这些平滑肌细胞用磷酸化的-HSP27 cDNA转染。