双极性躁郁症（Bipolar Disorder）的人诱导多能干细胞模型的建立和神经病理研究

Bipolar Disorder (BD) is a complex heritable neuropsychiatric disorder that affects more than 1% of the population worldwide. Patients with BD usually exhibit intermittent episodes of mania and depression, of which 20% would choose to suicide if without effective treatment. Hence, BD has been ranked by the WHO as a top disorder of lost life among all medical diseases. However, the roles and causation of the discovered physiological changes in BD brains are too complex to exactly determine the pathology of the disease. This has been proved by the fact that none of the current BD animal models can recapitulate both manic and depressive phenotypes of BD simultaneously. Therefore, developing an accurate and powerful biological model has been a challenge for research into BD. Lately, the development of induced pluripotent human stem cell (iPSC) technology, which is based on the revolutionary reprogramming of adult somatic cells, provides almost an unlimited number of stem cells for disease research (Thompson, Yamanaka). Since its naissance, the iPSC models of a number of inherited neuronal disorders have been established. Many of these models succeeded in recapitulating the neuropathological phenotypes or genetic deficiencies detected from patients or analogue animals, indicating that the cell lines reprogrammed from the somatic cells of humans carry the natural genetic background of the patients. Hence, this approach is particularly useful for the study of complex multigenic diseases such as BD. In this project, we will develop a human iPSC model of BD through reprogramming fibroblasts of BD patients, and differentiate these iPSCs into hippocampal dentate gyrus granule cells through a neural progenitor cell (NPC) pattern. Furthermore, using electrophysiological recording and somatic Ca2+ imaging techniques, as well as mitochondrial functional assays, we will investigate the phenotype of the disease at the cellular level. Finally, using the RNA-seq method, we will probe for genes involved in the pathogenesis of BD and pharmacological mechanisms of clinical drugs. Our study will generate a powerful tool for research into BD and make a significant contribution to understanding the etiology of this disease.

双极性躁郁症是一种复杂的遗传性神经心理学疾病，患者不断交替经历躁狂与抑郁两种相反的极端情绪状态。该病引起的自杀率极高，因此被世界卫生组织列为六大最致命疾病之一。然而，关于躁郁症的发病机制的研究长期以来进展极为缓慢。这主要是因为这类复杂疾病常由多个基因和信号通路的缺陷组成，各部分变化又相互影响，因而目前针对单一基因或通路的生物模型和方法难以满足研究需要。近年来，诱导多能干细胞模型被广泛用于神经疾病的机理研究，并在某些简单疾病方面已进入临床药物筛选的实用阶段。鉴于该模型能够忠实表现疾病的遗传信息，其在研究复杂的多基因疾病包括双极性躁郁症方面，具有不可比拟的优势。在本项目中，我们将建立躁郁症病人诱导多能干细胞系，并将其分化为与躁郁症发病有关的海马齿状回神经元。进一步，利用多种技术手段，我们将系统研究躁郁症在神经元水平上的病理变化。最终，我们将进行转录组测序以分析该病的可能致病基因群和信号通路。

双极性躁郁症的发病机制的研究长期以来进展缓慢，这主要是因为这类复杂疾病常由多个基因和信号通路的缺陷组成，各部分变化又相互影响，因而目前针对单一基因或通路的动物模型和方法难以满足研究需要。近来年,诱导多能干细胞技术在精神疾病研究领域得到了广泛应用，由病人的上皮细胞重编程获取的干细胞分化成的神经元被认为能够较好地携带病症的遗传缺陷。本项目组构建了躁郁症病人的诱导多能干细胞模型和相应的海马区齿状回颗粒神经元分化模型。利用转录组测序、神经电生理和活细胞荧光成像等多种前沿技术手段，本项目组系统地研究了躁郁症在神经元水平上的基因表达谱异常和病理变化，发现海马神经元超兴奋性是躁郁症在细胞水平的缺陷表型，这一缺陷很可能是信号激酶、钠钾离子通道和线粒体功能等多个系统的基因过度表达引起的。并且，本项目组系统分析了临床药物锂盐治疗躁郁症的可能机理。这一成果于2015年发表于《Nature》。为进一步在小鼠模型中验证从病人干细胞中获得的与发病相关的可疑基因，本项目组对传统的CRISPR/Cas9技术针对神经系统进行改良，开发出了神经系统特异性的CRISPR interference技术，能够快速构建小鼠脑部多重基因条件性敲低的模型，大大加快了精神疾病发病基因筛选的效率。这一成果于2018年发表于《Nature Neuroscience》。本项目取得一系列重要成果，共发表论文四篇，并启动了对CRISPRi技术的知识产权专利的申请工作，目前正在国家专利和国际PCT专利申请过程中。

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