Ryanodine受体在脊髓白质损伤中的作用及机制研究

Spinal cord injury causes catastrophic consequences and white matter damage is one of key factors to block the motor function recovery of paraplegic patients. Main pathological changes of white matter injury include oligodendrocytic cell death and axonal degeneration. Glutamate receptor-mediated Ca2+ overload from extracellular sources was considered to play a major role in white matter injury. However, in many cases Ca2+ overload and axonal injury still occurs in spite of removal of extracellular Ca2+. Our latest findings showed that the expression of ryanodine receptor (RyR), the most important channel regulating intracellular Ca2+ release from endoplasmic reticulum, was significantly increased and located in oligodendrocytes and axons and mediated the accumulation of intracellular Ca2+ after spinal cord injury, strongly indicating that RyR-mediated Ca2+ release from intracellular stores may be involved in Ca2+ overload and subsequently in spinal cord white matter injury. As a result, this project is designed in order to explore the effects and underlying mechanisms of RyR on spinal cord white matter injury. Using traumatic and ischemic animal models, we will investigate the relationship between the change in spatial-temporal expression of RyR and the extent of spinal cord injury. Using the methods such as pharmacologic intervention and viral vector infection, we will explore the effects of RyR on white matter injury and underlying mechanisms in vivo. In a hypoxia culture system, using RNAi interference and co-immunoprecipitation, we will investigate the effects of RyR on oligodendrocytes and the key molecules regulating RyR functions. The expected results will provide an experimental evidence for a novel mechanisms underlying white matter injury, therapeutic targets and new strategy of prevention and treatment for spinal cord injury.

脊髓损伤后髓内白质的变性死亡是截瘫患者运动功能难以恢复的根本原因之一。少突胶质细胞死亡和神经元轴突变性是白质损伤的重要病理改变。以往认为，谷氨酸受体介导的细胞外钙内流引发的钙超载是白质损伤的关键环节。然而新近研究证明，当去除胞外钙后，胞内钙超载依然存在。我们也发现，控制胞内钙池钙释放的一个重要通道- - RyR受体在脊髓损伤后表达明显增高且定位于少突胶质细胞，并参与胞内钙浓度的升高，强烈提示RyR介导的胞内钙释放亦可引发钙超载参与脊髓白质损伤的发生发展。为此，本课题拟在动物脊髓损伤模型上研究RyR表达时空变化与脊髓白质损伤的关系；利用药物干预、病毒感染等方法研究RyR对白质损伤及对运动功能的作用；在体外缺氧模型上，通过RNAi干扰、免疫共沉淀等方法探讨RyR对少突胶质细胞的作用和调控RyR功能的关键分子机制，以揭示RyR的新功能，为阐明脊髓创伤的白质损伤机制、治疗靶点及防治新策略提供实验依据。

脊髓损伤（spinal cord injury，SCI）后髓内白质的变性死亡是截瘫患者运动功能难以恢复的重要原因之一。少突胶质细胞死亡和轴突变性是白质损伤的重要病理改变，但发生机制尚未完全阐明。我们课题组在前期预实验中发现，控制胞内钙池钙释放的一个重要通道——Ryanodine受体（RyR）在脊髓损伤后表达明显增高，且定位于少突胶质细胞和轴突，强烈提示RyR介导的胞内钙释放可能参与脊髓白质损伤的发生发展。为此，本课题在大鼠SCI损伤模型上，研究RyR不同亚型在脊髓白质中的细胞定位和SCI后的表达变化，结果显示RyRs表达于脊髓白质的少突胶质细胞和神经纤维，SCI损伤后其表达水平显著增高，提示RyRs可能参与SCI后脊髓白质损伤的过程；利用药物干预的方法，探讨RyR对脊髓白质损伤的作用，结果表明增强RyR活性可加重大鼠SCI后脊髓白质的损伤程度，减弱RyR活性则可减轻白质损伤，提示RyR在SCI后脊髓白质损伤中发挥重要作用；通过动物行为学试验研究RyR对大鼠运动功能恢复的影响，结果证实改变RyR活性可影响SCI后大鼠的运动功能评分，抑制RyR可促进SCI后动物后肢运动功能的恢复。本研究为阐明脊髓白质损伤机制和防治策略提供实验依据。