利用诱导多能干细胞研究丙戊酸诱发Alpers-Huttenlocher综合征肝毒性的机理

发布时间：2019-06-14 23:28

【摘要】：丙戊酸钠(VPA)是目前在世界范围内使用最广泛的一线抗惊厥药物和定期用于偏头痛治疗、双相性精神障碍和慢性头痛的治疗药物,并且经常也会拿来作为辅助的化疗,但是VPA的使用会造成严重肝损伤的副作用。Alpers综合征(AHS),是一种由于线粒体DNA聚合酶POLG基因突变造成的遗传性线粒体遗传性疾病。Alpers综合征最显著的一个肝病特征是对于在幼儿时期发生的VPA诱导的肝毒性具有很高的风险。然而,这种高风险的肝毒性发生的机制是什么一直还不清楚,因此本文试图利用体外分化建立的疾病模型来探索其中的原因。在本研究中,我们将两例AHS病人的成纤维细胞诱导成为病人特异性的诱导多能性细胞,然后在用无血清体系下将它们的多能干细胞分化成病人来源特异性肝细胞(AHS iPSCs-Hep)。两个病人的分化的肝细胞与正常的对照细胞相比,都对VPA诱导的肝毒性非常敏感,产生凋亡。并且,细胞凋亡是依赖线粒体途径的,因为凋亡蛋白caspase-9和caspase-3都被激活,而且细胞色素c也被释放出来。与此同时,值得注意的是,AHS病人肝细胞中线粒体内保持线粒体嵴紧密和保护细胞色素c不被释放出来的OPA1复合物的量也比正常对照的要少。进一步研究发现,AHS病人肝细胞的线粒体DNA聚合酶POLG的表达量和线粒体DNA的含量比正常对照细胞的都要少,而且,AHS病人肝细胞ATP的产生能力也较正常对照的要弱。AHS iPSCs分化得到肝细胞AHS iPSCs-Hep其线粒体的结构也不正常,嵴变得少儿膨大,甚至有些线粒体缺少嵴。在研究VPA诱导AHS iPSCs-Hep凋亡敏感性机制时,我们发现,一种由于线粒体超氧化物的量瞬时增加而产生的超氧炫,其产生的频率比正常对照细胞要高,而这种超氧炫的产生本质上是由线粒体通透性转换孔(mPTP)的开放引起的。同时,当我们加入mPTP抑制剂时,AHS iPSCs-Hep对于VPA诱导的肝毒性的敏感性下降到正常对照的水平。本研究的这些结果说明如果把线粒体mPTP作为药物靶点设计药物,很有可能会防止AHS病人对于VPA诱导的肝毒性的发生。此外,曾报道过用于治疗VPA诱导的肝毒性的药物乙酰胆碱carnitine和N-acetylcysteine (NAC),在本研究的疾病模型中同样可以降低AHS病人对VPA诱导的肝毒性的敏感性。总之通过AHS病人成纤维细胞来源的iPSCs建立的AHS病人的肝疾病模型的研究,揭示了AHS病人肝细胞对VPA诱导的肝毒性的机制,证明其是因为AHS病人肝细胞线粒体mPTP的开放引发的依赖线粒体途径的细胞凋亡。本研究是首次使用iPSCs建立的遗传性线粒体疾病的毒理学模型,这一模型可以为筛选治疗药物提供新的评估方法。
[Abstract]:Sodium valproate (VPA) is currently the most widely used first-line anticonvulsant in the world and regularly used in migraine treatment, biphasic mental disorder and chronic headache, and is often used as an auxiliary chemotherapy, but the use of VPA can cause side effects of severe liver injury. Alpers syndrome (AHS), One of the most significant characteristics of liver disease in Alpers syndrome is that it has a high risk for VPA-induced hepatotoxicity in early childhood. However, it has not been clear what the mechanism of this high risk hepatotoxicity is, so this paper attempts to explore the causes by using the disease model established by differentiation in vitro. In this study, the fibroblasts of two patients with AHS were induced into patient-specific induced pluripotency cells, and then their pluripotent stem cells were differentiated into patient-specific hepatocytes (AHS iPSCs-Hep) in a serum-free system. Compared with the normal control cells, the differentiated hepatocytes of both patients were very sensitive to VPA-induced hepatotoxicity and produced apoptosis. Moreover, apoptosis depends on the mitochondrial pathway because both apoptosis proteins caspase-9 and caspase-3 are activated and cytochrome c is released. At the same time, it is worth noting that the amount of OPA1 complex in the hepatocytes of AHS patients that keeps the crest of mitochondria tight and protects the release of cytochrome c from release is also less than that of the normal control group. It was found that the expression of DNA polymerase POLG and the content of mitochondrial DNA in hepatocytes of AHS patients were lower than those of normal control cells, and the production ability of ATP in hepatocytes of AHS patients was weaker than that of normal controls. The structure of hepatocytes AHS iPSCs-Hep was not normal, and the cristae became enlarged in children, and even some mitochondria lacked cristae. In studying the sensitivity mechanism of AHS iPSCs-Hep apoptosis induced by VPA, we found that a kind of hyperoxia produced by the instantaneous increase of the amount of mtDNA superoxide produced higher frequency than that of normal control cells, and the production of this type of hyperoxia was essentially caused by the opening of (mPTP) in the permeability transition pore of mitochondria. At the same time, when we added mPTP inhibitor, the sensitivity of AHS iPSCs-Hep to VPA-induced hepatotoxicity decreased to the level of normal control. These results suggest that if mtmPTP is used as a drug target to design drugs, it is possible to prevent the hepatotoxicity induced by VPA in AHS patients. In addition, it has been reported that acetylcholine carnitine and N-acetylcysteine (NAC), which are used to treat VPA-induced hepatotoxicity, can also reduce the sensitivity of AHS patients to VPA-induced hepatotoxicity in the disease model of this study. In a word, the liver disease model of AHS patients established by iPSCs from fibroblasts of AHS patients revealed the mechanism of hepatotoxicity induced by VPA in AHS patients, which proved that the mechanism of hepatotoxicity induced by VPA in AHS patients was due to the apoptosis induced by the opening of mitochondrial mPTP in AHS patients. This study is the first time that iPSCs has been used to establish a toxicological model of hereditary mitochondrial disease, which can provide a new evaluation method for screening therapeutic drugs.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R96

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