脑缺血再灌注中靶向调控Neuritin的转录因子及分子机制研究

Neuritin, a novel members of the neurotrophin family, can promote neurite outgrowth, adjust the formation of synaptic circuits, while without the side effects of neuralgia pain, these significant characteristics make it become a new potential targets for nerve repair and regeneration. However, the genetic engineering technology is difficult to obtain full-length Neuritin protein, and the method and route of administration still exist problems, so the development of small molecule drug in the regulation of Neuritin endogenous expression become a preferred choice. Transcription regulation is an important part of the gene expression and regulation, but the molecular mechanism of Neuritin transcription regulation is still unclear. Accordingly, our research is aimed to investigate the specific transcription factors that can target regulate Neuritin gene in transcriptional level by using transient global cerebral ischemia and reperfusion rat model. We will exploit a combined bioinformatics and biochemical approaches for multilayer screening, then further validate in vivo and in vitro, and finally confirm in cellular level precisely. The outcome of this study will illustrate a regulation map for these transcription factors-binding sites implicated in establishing transcriptional patterns of Neuritin gene expression. The proposal may further elucidate the molecular mechanism of Neuritin transcriptional level in neural repair and nerve regeneration, provide theoretic foundation of development attractive drugs for treatment these kinds of disease, and offer a new strategy to address the unmet medical need in the patient population.

神经营养因子家族新成员—Neuritin，能促进神经突起生长，调节突触回路的形成，却无致神经痛的副作用，成为神经再生修复领域的新的潜在靶点。然而，目前的基因工程技术很难获得Neuritin全长蛋白，且给药方式和途径存在难题，因此开发小分子调控Neuritin内源性表达的药物成为优先选择。转录水平的调控是基因表达调控的重要环节，但国际学界对Neuritin转录调控的分子机制仍不明确。基于此，本项目从大鼠全脑短暂性缺血再灌注模型入手，利用生物信息学与分子生物学实验相结合，通过多层筛选、体内与体外实验及细胞水平的验证，深掘与Neuritin发生作用的特定转录因子，阐明这些转录因子与Neuritin基因靶向调控的关系，揭示神经营养因子Neuritin转录水平调控的分子机制，为开发治疗神经损伤的药物提供理论依据。

Neuritin是一种在神经损伤后具有修复功能的神经营养因子，但对其转录调控的分子机制仍未见报道。本项目首先通过生物信息学及实验方法相结合，筛选及鉴定大鼠Neuritin 基因启动子区。在此基础上使用多款生物信息学预测软件交集筛选与大鼠Neuritin 基因启动子主要调控区结合的转录因子，利用双荧光素酶报告基因技术和绿色荧光蛋白表达检测技术，在细胞水平确定转录因子主要调控区的转录活性。最后通过大鼠全脑短暂性缺血再灌注模型，应用DNA pull down和质谱分析技术，确定转录因子CREB 和AP-1 家族对大鼠Neuritin 基因启动子主要调控区的调控作用。这些研究成果揭示了神经营养因子Neuritin转录水平调控的分子机制，为开发治疗神经损伤的小分子药物药物提供理论依据。