APOPTOSIS IN SKELETAL MUSCLE FOLLOWING BURN INJURY

烧伤后骨骼肌细胞凋亡

• 批准号: 6363354
• 负责人: Jeevendra Martyn
• 金额: $ 33.72万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2004-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6363354
• 关键词: BCL2 gene /protein; RNase protection assay; apoptosis; biological signal transduction; burns; cysteine endopeptidases; disease /disorder etiology; enzyme linked immunosorbent assay; flow cytometry; gel electrophoresis; gene targeting; genetically modified animals; immunoprecipitation; laboratory rat; muscle function; muscle strength; myofibrils; neuromuscular disorder; phosphatidylinositol 3 kinase; polymerase chain reaction; scintillation counter; striated muscles; thin layer chromatography; western blottings

Burn injury, with and without sepsis, is associated with many functional and metabolic aberrations. In skeletal muscle, the important functional change is muscle weakness resulting in hypoventilation, dependence on respirators, and decreased mobilization. The neuromuscular dysfunction and muscle weakness associated with burns prolongs hospital stay, increases cost, morbidity and mortality. The long-term goals of these studies are, therefore, to characterize the etiology of muscle weakness and design strategies to improve tension-generating capacity of muscle in humans. Based on convincing preliminary data, the hypotheses tested in the present proposal is that apoptosis or programmed cell death occurs in skeletal muscle, at sites distant and immediately local to burn, with activation of pro-apoptotic signaling pathways. The goals of the present studies, using rats, are to: (1) Confirm and study the evolution of apoptosis in skeletal muscle at local and distant sites from burn, and in different fiber types (slow vs. fast twitch muscles). (2) Characterize the putative signaling pathways leading to apoptosis, previously documented only in in vitro systems. (3) Effectively inhibit some of the activated pro-apoptotic pathways and decrease muscle wasting and muscle weakness. Apoptosis will be confirmed by three independent methods- the ladder, TUNEL and ELISA assays. The cell membrane and downstream signaling molecules modulating apoptosis (e.g., TNFalpha, Fas, FasL, phosphatidylinositol 3-kinase, ceramide, BCL-2, BCL-X and caspases) will be assessed for expression and/or activity by using molecular pharmacological or biochemical techniques. The importance of certain signaling molecules will be confirmed by the use of knock out or transgenic (TNF, lpr or Fas, ceramide, BC12, and caspase) mice. Specific exogenous modulators (e.g., IGF-1 and caspase inhibitors) of some of the apoptotic signaling molecules will be used in vivo to attenuate muscle wasting due to apoptosis. These "reversal of apoptosis" studies will be performed in conjunction with protein turnover and functional studies to test the efficacy of these drugs to reverse the neuromuscular dysfunction of burns. The proposed studies will, therefore, delineate the neuromuscular dysfunction associated with burns, the component contributed to by apoptosis, and the potential for attenuation of both the apoptosis and the neuromuscular dysfunction by pharmacotherapy. Information obtained from the rat studies will provide a scientific basis and rationale for therapeutic maneuvers to prevent and/or rectify neuromuscular complications of burns or critical illness in humans.

烧伤合并或不合并脓毒症，与许多功能和代谢异常有关。在骨骼肌中，重要的功能变化是肌肉无力，导致换气不足，对呼吸机的依赖，以及活动减少。与烧伤相关的神经肌肉功能障碍和肌肉无力延长了住院时间，增加了费用，增加了发病率和死亡率。因此，这些研究的长期目标是确定肌肉无力的病因，并设计策略来提高人类肌肉的张力产生能力。基于令人信服的初步数据，本方案中检验的假设是，骨骼肌发生细胞凋亡或程序性细胞死亡，发生在距离烧伤较远和直接位于烧伤局部的部位，并激活促凋亡的信号通路。本研究以大鼠为研究对象，目的是：(1)证实和研究烧伤后局部和远端骨骼肌以及不同纤维类型(慢肌和快肌)中细胞凋亡的演变。(2)鉴定以前仅在体外系统中报道的导致细胞凋亡的可能的信号通路。(3)有效抑制部分激活的促细胞凋亡途径，减少肌肉萎缩和肌肉无力。细胞凋亡的确认将通过三种独立的方法--梯状条带、TUNEL和ELISA法。调控细胞凋亡的细胞膜和下游信号分子(例如，TNFpha、Fas、FasL、磷脂酰肌醇3-激酶、神经酰胺、bcl2、bclX和caspase)将通过分子药理学或生化技术来评估其表达和/或活性。某些信号分子的重要性将通过使用敲除或转基因(肿瘤坏死因子、LPR或Fas、神经酰胺、BC12和半胱氨酸酶)小鼠来证实。一些凋亡信号分子的特定外源调节剂(如IGF-1和caspase抑制剂)将在体内用于减轻因细胞凋亡而导致的肌肉萎缩。这些“细胞凋亡逆转”研究将与蛋白质周转和功能研究一起进行，以测试这些药物逆转烧伤神经肌肉功能障碍的疗效。因此，拟议的研究将描绘与烧伤相关的神经肌肉功能障碍，细胞凋亡所致的成分，以及通过药物治疗减轻细胞凋亡和神经肌肉功能障碍的可能性。从老鼠研究中获得的信息将为预防和/或纠正人类烧伤或危重疾病的神经肌肉并发症的治疗操作提供科学基础和理论基础。