以凋亡诱导因子为靶点防治新生儿脑损伤的研究

Despite major improvements in maternal care, obstetrics and neonatology, the number of newborn children suffering from brain injuries occurring around birth is increasing. One reason for this is that more children born much too early and survive. Neonatal brain injury is a frequent cause of cerebral palsy, which is the most common cause of severe disability in children. Considerable efforts have been focused on treatments for neonatal brain injury; however, to date, most have been unsuccessful. The current therapy for preterm brain injury is mainly supportive. There is an urgent need for developing neuroprotective approaches for this condition. Apoptosis plays a far greater role in the immature brain than in the adult brain. Apoptosis-Inducing Factor (AIF) is very important for neuronal cell death which could be a targeted protective strategy for limiting neonatal brain injury. We have identified a brain-specific form of this protein, AIF2. We have also identified a protein not previously studied in the brain, Mia40, which binds to AIF. In this proposal, we will use AIF conditional knock out mice as well as Mia40 heterozygous mice and their wild type controls to produce neonatal hypoxia ischemia brain injury model. The effect of AIF2 or Mia40 on mitochondrial respiration, neural stem / progenitor cells proliferation, oxidative stress and neuronal degeneration will be investigated in the physiological and pathological conditions. The effect on neuronal cell death and brain injury as well as brain function will be evaluated after hypoxia ischemia. The synthetic peptide based on the structure of AIF will be tested on glutamate induced HT-22 cell culture system. This study will enable us to understand how AIF works and how we can best interfere to reduce brain injury or promote brain functional recovery in the immature brain after insult.

应用条件性基因敲除或者基因突变小鼠的缺氧缺血脑损伤模型，以凋亡诱导因子及辅因子为研究靶点，采用免疫组织化学、细胞生物学、生物物理学、分子生物学以及动物神经行为测定等方法，结合荧光标记、光共聚焦显微镜、无偏差体视显微镜及线粒体呼吸功能测定等技术，探讨凋亡诱导因子及辅因子尤其是脑特异性凋亡诱导因子的亚型、线粒体膜腔隙转运蛋白40与脑发育和脑损伤的关系，并根据凋亡诱导因子与辅因子亲环蛋白A的表面分子结构合成能与凋亡诱导因子或者亲环蛋白A竞争性结合的小分子肽，进行抑制神经细胞死亡的体内外实验，为新生儿脑损伤的防治提供理论基础和新的措施。

新生儿脑损伤是新生儿期最常见、最严重的疾病之一，目前仍然缺乏有效的治疗措施，是导致新生儿时期致死、致残的常见原因。线粒体膜强隙包含大量的与细胞存亡相关的蛋白分子，是调控细胞存亡的重要细胞器。本项目主要采用线粒体与细胞存亡相关的蛋白分子转基因幼年小鼠脑损伤模型，探讨不同蛋白分子在病理状态下与神经细胞死亡的关系以及信号传导通路，寻找脑损伤的防治措施。制备了脑特异性凋亡诱导因子(AIF2)基因敲除小鼠，发现神经元特异性AIF2基因敲除对小鼠的生长发育、存活、生殖功能以及神经元线粒体的结构和功能无明显影响。Mia40是线粒体膜腔隙蛋白具有调节线粒体膜腔隙蛋白转运功能，我们发现凋亡诱导因子下调能导致Mia40蛋白水平的的下降，而Mia40的下调能导致类似于凋亡诱导因子下调的线粒体呼吸功能缺陷，采用Mia40下调的新生鼠脑损伤模型证实Mia40下调能减轻神经细胞死亡以及缺氧缺血性脑损伤程度。对自吞噬基因7(Atg7)条件性敲除与脑组织损伤的关系进行研究，发现Atg7在神经元中选择性敲除后能减少损伤引起的凋亡诱导因子从线粒体释放以及神经细胞死亡对缺氧缺血性脑损伤有保护作用，能够抑制小胶质细胞激活以及炎症因子的释放，但是对线粒体裂变及聚变功能、线粒体的生物合成功能无影响。应用对线粒体代谢具有调节作用的二氯乙酸对脑损伤模型进行研究， 发现脑损伤后给予二氯乙酸能改善线粒体的生物合成，抑制自吞噬和细胞凋亡，明显减轻脑损伤的程度。采用临床相关的延迟性给药的方案对脑损伤的影响进行探讨，发现半胱天冬酶的抑制剂以及锂盐对未成熟脑损伤均有神经保护作用，其机制可能与抑制炎症反应有关。研究结果显示，以线粒体为靶点通过调节线粒体关键蛋白分子或代谢功能对脑损伤具有防治作用，为新生儿脑损伤的防治提供了理论基础。