冷休克蛋白RBM3基因的低温诱导机制及其模拟亚低温用于脊髓损伤治疗

Mild hypothermia is a well-established therapeutic tool to alleviate neural injures. Because of its adverse effects, it is urgently required to duplicate cooling's protection pharmacologically. The previous studies by us and others strongly suggest that cold shock protein RBM3 (RNA binding motif protein 3) can mimics mild hypothermia to protect neural cells from apoptosis induced by various insults, supporting the hypothesis that inducing RBM3 specifically in injured brain areas may be more beneficial than therapeutic hypothermia. However, there is no protocol available for RBM3-specific induction, due to poor understanding on its gene expression control. Meanwhile, the effects of RBM3-based therapy on recovery of neural injury remain unclear. In the present study, we aim to determine hypothermia-related signaling pathways responsible for RBM3 induction, using high-throughout phosphorylation antibody chips. Next, techniques including LC-MS/MS, EMSA, ChIP and promoter reporter assays will be employed, in order to isolate and identify the specific transcription factors regulating RBM3 expression under mild hypothermic conditions and cold-responsive elements (CRE) within RBM3 gene promoter. Based on that, the therapeutic effects of recombinant lentiviruses expressing RBM3 and the transcriptional factor genes on spinal cord injury (SCI) in rats will be evaluated. This study is to elucidate the mechanisms underlying gene expression control of RBM3 by mild hypothermia, and the findings will shed a light on using RBM3-targeted drugs to treat neural injuries clinically.

亚低温是目前临床上公认用来减轻神经损伤的有效手段，但其副作用大，因此迫切需要寻找新的方法来模拟或替代亚低温疗法。我们前期的研究发现，冷休克蛋白RBM3可以模拟亚低温，保护多种应激因素诱发的神经细胞凋亡，提示可通过RBM3的特异性诱导替代低温治疗。而RBM3基因的表达调控机制，以及RBM3特异性表达（或诱导）对神经损伤的实际疗效均不清楚。本课题拟利用磷酸化抗体芯片技术，明确参与RBM3基因诱导的冷应激相关信号通路；借助LC-MS/MS、EMSA、ChIP及启动子报告实验等技术，分离并验证特异性调控RBM3表达的转录因子及RBM3启动子上的冷应答元件；并利用大鼠脊髓损伤模型，评价RBM3及其转录因子基因的重组慢病毒对脊髓损伤的实际疗效。其结果将揭示低温对冷休克蛋白RBM3基因的具体调控机制，为RBM3特异性靶向药物应用于神经损伤的临床治疗提供理论依据。

亚低温是临床公认的减轻神经损伤的有效手段，但其副作用大，因此寻找手段模拟亚低温保护，成为目前临床上治疗神经损伤亟待解决的问题。前期的研究发现，冷休克蛋白RBM3可以模拟亚低温保护神经损伤，而RBM3基因的表达调控机制以及RBM3特异性表达（或诱导）对神经损伤的疗效均不清楚。本课题采用了多种神经细胞损伤模型，研究RBM3的神经保护作用，发现RBM3可以有效抵抗MPP+或紫外（UV）辐射诱导的神经细胞凋亡及鱼藤酮（ROT）诱导的神经毒性，并分析了RBM3保护神经细胞凋亡的机制；利用一种新型的非放射性的方法（SUnSET）研究了RBM3对细胞总蛋白合成的影响，并对RBM3潜在的下游靶基因RTN3以及YAP1的表达进行了分析，发现RBM3在蛋白质翻译水平对RTN3存在调控作用，而在mRNA转录水平对YAP1发挥调控作用；进一步研究RBM3在亚低温条件下的转录调控机制，发现FAK/Src通过NK-κB参与亚低温对RBM3基因的转录调控，并初步研究了FAK/Src信号的生物学意义，分析了其对ROT诱导的神经毒性的保护作用。本项目揭示了低温对冷休克蛋白RBM3基因表达的具体调控机制，为RBM3特异性靶向药物应用于神经损伤的治疗提供了一定的理论依据。