利用干细胞进行丙戊酸诱发Alpers-Huttenlocher综合征肝毒性的机理研究

Alpers-Huttenlocher syndrome (AHS) is a neurometabolic disorder caused by mutations in mitochondrial DNA polymerase gamma (POLG). Diagnosis of AHS is based on the clinical triad of intractable epilepsy, and characteristic liver disease. Valproic acid is widely used to treat epilepsy, but potentially causes idiosyncratic liver injury. AHS is associated with an increased risk of developing fatal valproic acid hepatotoxicity, and approximately one third of AHS patients develop liver failure within 3 months of exposure to valproic acid. However, the mechanistic link of this clinical mystery remains unknown. We have found that AHS hepatocytes are more sensitive to valproic acid-induced apoptosis, which is through mitochondrial pathway (Hepatology, 2015). In this project, we would study the mechanism and drug screening of valproic acid-induced hepatotoxicity in Alpers-Huttenlocher syndrome in an induced pluripotent stem cell (iPSC) model. The objects include: (1) Mitochondrial mechanism of AHS hepatocytes sensitivity to valproic acid induced apoptosis; (2) Valproic acid metabolic mechanism of AHS hepatocytes sensitivity to valproic acid induced apoptosis; (3) Gene correction and drug screening during AHS iPSCs differentiation to hepatocytes. Our research will elucidate the mechanism of valproic acid-induced hepatotoxicity in AHS, enable the evaluation of drugs for the therapeutic target, and provide a hepatotoxicity study model by using iPSCs.

Alpers-Huttenlocher综合征（AHS）是线粒体DNA聚合酶POLG突变引起的遗传疾病，临床常有难治性癫痫和肝病等症状。丙戊酸是一种广谱抗癫痫药物，但对AHS病人进行治疗时，经常会导致病人急性肝衰竭（约1/3的病人服用3个月发生肝衰竭），机制一直不明。我们已经发现AHS病人肝细胞对丙戊酸诱导凋亡敏感，并通过线粒体途径凋亡（Hepatology, 2015）。本项目将通过诱导多能干细胞（iPS）技术建立疾病模型，研究丙戊酸诱发AHS特异肝毒性的内因外因，及基于机理的药物筛选，包括：（1）AHS病人肝细胞对丙戊酸诱导凋亡敏感的线粒体机制；（2）AHS病人肝细胞对丙戊酸诱导凋亡敏感的丙戊酸代谢机制；（3）利用AHS病人iPS细胞分化为肝细胞进行基因修复与药物筛选。本研究将阐明丙戊酸诱发AHS特异肝毒性的机理，建立相应的候选治疗策略，可望为肝脏内药物的毒理研究提供干细胞研究的范例。

项目背景：Alpers-Huttenlocher综合征（AHS）是线粒体DNA聚合酶POLG突变引起的遗传疾病，临床常有难治性癫痫和肝病等症状。丙戊酸是一种广谱抗癫痫药物，但对AHS病人进行治疗时，经常会导致病人急性肝衰竭，机制一直不明。我们已经发现AHS病人肝细胞对丙戊酸诱导凋亡敏感，并通过线粒体途径凋亡（Hepatology,2015）。.研究内容：本项目将通过IPS技术建立疾病模型，研究丙戊酸诱发AHS特异肝毒性的内因外因，及基于机理的药物筛选，包括：（1）AHS病人肝细胞对丙戊酸诱导凋亡敏感的线粒体机制；（2）AHS病人肝细胞对丙戊酸诱导凋亡敏感的丙戊酸代谢机制；（3）利用AHS病人iPS细胞分化为肝细胞进行基因修复与药物筛选。.重要结果：.1） AHS病人肝细胞对丙戊酸诱导凋亡敏感的线粒体机制。.1.1）电镜显示 AHS iPSCs-Hep 线粒体形态异常；.1.2）AHS iPSCs-Hep 线粒体 DNA 聚合酶表达情况；.1.3）AHS iPSCs-Hep 线粒体 DNA 的含量变化；.1.4）AHS iPSCs-Hep 产生 ATP 的功能异常；.1.5）AHS iPSCs-Hep 在体外培养用VPA处理更容易凋亡；.1.6）VPA 导致AHS iPSCs-Hep 的凋亡是通过线粒体途径的；.1.7）线粒体的超氧炫在AHSiPSCs-Hep线粒体中更加频繁。.2）AHS病人肝细胞对丙戊酸诱导凋亡敏感的丙戊酸代谢机制；.2.1）AHS iPSCs-Hep线粒体通透转运孔道（mPTP）开放频率增加；.2.2）mPTP的抑制剂可以缓解 AHS iPSCs-Hep对 VPA 引起的凋亡的敏感性；.2.3）AHS iPSCs-Hep线粒体中可能组成mPTP的复合物的组成和功能变化。.3）利用AHS病人iPS细胞分化为肝细胞进行基因修复与药物筛选；.3.1）利用AHS病人iPS细胞分化为肝细胞进行基因修复；.3.2）利用AHS病人iPS细胞分化为肝细胞进行药物筛选。.科学意义：本研究建立了体外AHS疾病模型，通过得到病人的iPSCs细胞源性肝细胞来研究VPA诱发的肝毒性，本研究还是首次利用患者来源的iPSCs细胞进行遗传性线粒体疾病的毒理性研究，并且可以为治疗药物提供有效评估的手段。