NRSF-CART在缺血性脑损伤疾病修复中的作用研究

Cocaine- and amphetamine-regulated transcript (CART) peptide plays a pivotal role in neuroprotection against ischemic stroke-related brain injury. We recently found that the transcriptional repressor neuron-restrictive silencer factor (NRSF, also known as REST) represses CART expression through direct binding to two NRSE elements in the CART promoter and intron 1. NRSF expression was increased significantly during oxygen-glucose deprivation which mimicks ischemic and hypoxia injury. The elevated NRSF prevented the neuroprotective role of CART. These data suggest that NRSF is an important signal transducer upon the neuronal damamge and targeting NRSF by RNAi-mediated depletion might be an useful strategy to upregulate the CART expression and then promote the brain injury repair. In this project, we will establish the oxygen-glucose deprivation which mimicks ischemic and hypoxia injury at both the cellular level (in human neuroblastoma SK-N-SH cells and primary cortical neuron) and the tissue level (brain slices). We will examine the expression changes of NRSF and CART during the injury occurrence and repair through qPCR and western blot analysis, and determine the apoptosis rate of neurons after oxygen-glucose deprivation through FACS and Tunel analysis. In addition, we will establish the middle cerebral artery occlusion (MCAO) suture model in Sprague-Dawley (SD) rats by in vivo observing the suture location with SR angiography. After injection of NRSF siRNA or forskolin into the lateral ventricle, the infarct volume and neuronal apoptosis will be detected with TTC and Tunel staining of brain samples. Taken together, this study will provide an useful target for the therapeutic interference of ischemic brain insult through NRSF and/or CART-based interference.

可卡因-苯丙胺调节转录肽CART在缺血性脑损伤中具有重要的保护作用。我们发现NRSF通过结合CART启动子、内含子的NRSE元件抑制CART转录，缺血、缺氧损伤诱导NRSF表达上调、进而阻断CART的神经保护功能，提示NRSF是神经损伤信号传递的重要因子，靶向敲低NRSF有望成为上调CART从而促进神经损伤修复的新策略。本项目拟在细胞水平（SK-N-SH细胞、皮质神经元）、组织水平（脑切片）建立模拟缺血、缺氧损伤的氧糖剥夺模型，利用实时定量PCR、蛋白质印迹等检测NRSF、CART的表达变化；通过流式分析、Tunel染色等检测神经细胞的凋亡率；在动物水平建立大鼠脑中动脉闭塞模型，通过侧脑室注射或递送系统对NRSF、CART进行干预，利用TTC、Tunel染色等测定脑梗塞面积和神经细胞凋亡率。该研究有助于阐明NRSF-CART轴在缺血性损伤及修复中的作用，为缺血性中风的治疗提供新的科学依据。

可卡因-苯丙胺调节转录肽CART在缺血性脑损伤中具有重要的保护作用。我们前期工作发现NRSF通过结合CART启动子、内含子的NRSE元件抑制CART转录，缺血、缺氧损伤诱导NRSF表达上调、进而阻断CART的神经保护功能，提示NRSF是神经损伤信号传递的重要因子，靶向敲低NRSF有望成为上调CART从而促进神经损伤修复的新策略。.本项目的主要研究内容包括：1. 在细胞水平，利用SK-N-SH细胞、原代分离的小鼠皮质神经元建立模拟缺血、缺氧神经损伤的OGD模型，分析转录负调控因子NRSF如何与其它的转录激活因子如CREB等竞争性调节CART基因的表达，探讨是否可以通过对NRSF、CART进行干预，从而减少缺血、缺氧导致损伤的神经元细胞凋亡、坏死数量；2. 在组织水平，制作大鼠脑切片，经OGD处理，分析NRSF、CART干预对脑片组织神经细胞生存率、凋亡率的影响；3. 在动物水平，建立大鼠MCAO模型，通过侧脑室注射或递送系统对NRSF、CART进行外源性干预，检测NRSF、CART干预前后大鼠脑梗塞面积、残存神经元细胞数、凋亡细胞数、血流动力学的变化，寻找缺血性脑损伤修复治疗性方案的实验依据。我们获得的重要结果和关键数据如下：不同水平的实验数据共同表明NRSF-NRSE可以有效地抑制cAMP/PKA/CREB正性调控作用的发挥，单纯由OGD引起的NRSF表达的上调并不足以抑制cAMP/PKA/CREB正性调控作用的发挥，需要有外源性的NRSF干预；外源性干涉NRSF可以使神经细胞生存率显著提高，添加Forskolin对CART进行联合干预可以进一步提高神经细胞的生存率。.本研究将有助于利用NRSF与CART基因的负调控关系，探讨在脑缺血、缺氧损伤状态下，如何通过干预NRSF、CART基因的表达为中风等疾病的治疗提供新的理论指导。