Developing An In Vitro Human Myobundle Model Of Rheumatoid Arthritis

摘要 类风湿关节炎(RA)是一种主要影响关节的慢性炎症性疾病。自体反应 免疫细胞进入关节和骨骼肌，滑膜发炎。 破坏软骨，导致疼痛和不适。从5到10年，RA的伤残率达到40% 在确诊后。除了疾病活动和疼痛外，肌肉力量对残疾也有重大影响 类风湿关节炎患者。骨骼肌显然会导致RA残疾，但其发生的机制是 不是很清楚。由于动物模型和细胞培养系统是人类疾病的不完美复制 并且不概括人类骨骼肌的生物学和生理学特征，我们建议使用 最近开发的工程化电响应、可收缩的人类肌肉组织(肌束)可 体外建立类风湿性关节炎模型，鉴定疾病表型特征。这些人类肌束显示出结构性的 天然骨骼肌的特征包括排列的结构，多核和条纹的肌纤维，以及 卫星电池池。在培养的四周后，它们会自发收缩，并对单次或高强度反应 伴有抽搐和强直性收缩的频率电刺激；肌束维持功能性乙酰胆碱 和β-肾上腺素能受体，并经历结构和功能成熟。成肌细胞的初步数据 来自类风湿性关节炎患者的研究表明，类风湿关节炎降低了肌纤维的形成能力，并显著 收缩力，即使在去除多余的成纤维细胞后也是如此。建立体外系统作为模型进行检验 对于骨骼肌疾病的治疗，我们将扩展我们的初步结果并检验假设 假说(1)工程骨骼肌肌束来源于类风湿关节炎患者的细胞 显示修复和分化能力降低以及部队生产减少；(2) 分化和功能是由于选择肌细胞因子和细胞因子的产生；以及(3)添加细胞因子和 RA骨骼肌肌纤维分泌的肌细胞因子可诱导健康成肌细胞的RA表型。在……里面 目的1，我们将扩展我们的初步结果，以表明工程骨骼肌肌束 复制疾病表型，表现出成熟度和产生力量的能力降低。 用年龄匹配的健康个体的成肌细胞制备的肌束将作为对照。结果 将与活检样本的免疫组织化学染色进行比较，以确定类风湿关节炎的细胞变化 肌肉。在目标2中，我们将使用一种公正的方法，测量从RA释放的蛋白质和细胞因子 肌束和应用统计模型确定RA表型特征之间的关系 在目标1中确定以及肌细胞因子和细胞因子的水平。在目标3中，我们将假设 RA肌肉异常分泌的肌细胞因子和细胞因子是该病表型的主要原因。

Abstract Rheumatoid arthritis (RA) is a chronic inflammatory disease that primarily affects the joints. Autoreactive immune cells enter articular joints as well as skeletal muscle, and inflammation of the synovial membrane damages the cartilage causing pain and discomfort. Disability rates from RA reach 40% from 5 to 10 years after diagnosis. In addition to disease activity and pain, muscle strength has a major impact on disability in RA patients. Skeletal muscle clearly contributes to RA disability, but the mechanisms by which this occurs are not well understood. Since animal models and cell culture systems are imperfect replicates of human disease and do not recapitulate biologic and physiologic features of human skeletal muscle, we propose to use a recently developed engineered electrically responsive, contractile human muscle tissues (myobundles) to model RA in vitro and identify features of the disease phenotype. These human myobundles exhibit structural hallmarks of native skeletal muscle including aligned architecture, multinucleated and striated myofibers, and a satellite cell pool. Over four weeks in culture, they contract spontaneously and respond to single or high frequency electrical stimuli with twitch and tetanic contractions; myobundles maintain functional acetylcholine and β-adrenergic receptors and undergo structural and functional maturation. Preliminary data with myoblasts derived from RA patients indicate that RA reduces the ability of myofibers to form and produce significant contractile force, even after removal of excess fibroblasts. To establish the in vitro system as a model to test therapies for the disease in skeletal muscle, we will extend our preliminary results and test the hypotheses hypotheses that (1) engineered skeletal muscle myobundles derived from cells of rheumatoid arthritis patients show reduced capacity for repair and differentiation and reduced force production; (2) the decrement in differentiation and function is due to select myokine and cytokine production; and (3) addition cytokines and myokines secreted by RA skeletal muscle myofibers can induce the RA phenotype in healthy myoblasts. In Aim 1, we will extend our preliminary results to show that the engineered skeletal muscle myobundles reproduce the disease phenotype and exhibit reduced maturation and capacity for force generation. Myobundles prepared with myoblasts from healthy aged-matched individuals will serve as controls. Results will be compared to immunohistochemical staining of biopsy samples to establish cellular changes in RA muscle. In Aim 2, we will use an unbiased approach and measure proteins and cytokines released from RA myobundles and apply statistical model to determine the relationship between features of RA phenotype identified in Aim 1 and the levels of myokines and cytokines. In Aim 3, we will that the hypothesis that the abnormal secretion of myokines and cytokines by RA muscle is responsible for the disease phenotype.