多种酰基辅酶A脱氢酶缺乏症iPS细胞的建立及PPARγ在肌肉细胞脂肪生成机制的研究

Multiple acyl-CoA dehydrogenase deficiency (MADD) is a hereditary metabolic myopathy characterized by impaired mitochondrial function and fatty acid beta oxidation dysfunction caused by mutations in ETF/ETFDH genes, resulting in a large number of lipid droplet formed in the muscle fibers. The mechanism of this disease was not fully understood. Our previous study found that the PPARγ expression levels of RR-MADD fibroblasts were increased, and fibroblasts could transdifferentiation to adipocyte and many lipid droplets formed under the adipogenic differentiation condition, while no lipid droplets formed in control group. The lipid droplets decreased significantly after PPARγ inhibitors treatment. We hypothesized that MADD not only have mitochondrial dysfunction, but also with the adipogenesis abnormalities, and PPARγ plays an important role in the. lipid deposition process. To discover the adipogenesis mechanism in MADD disease and provide new drug target for clinical treatment, we will generate iPS cells from MADD patients and then differentiate into skeletal muscle cells. During the myogenic differentiation, we will observe the formation of lipid droplets, measure the mitochondrial function, detect the expression of PPARγ and adipogenic genes. And further to learn the changes of lipid droplets and mitochondria function when treated to PPARγ inhibitors.

多种酰基辅酶A脱氢酶缺乏症（MADD）是由ETF/ETFDH基因突变导致线粒体功能受损，引起脂肪酸β氧化障碍的遗传性代谢性肌病，表现为肌纤维内脂滴异常增多。目前发病机制尚不完全清楚。我们前期研究发现MADD成纤维细胞PPARγ表达水平增高，在成脂诱导条件下转分化为脂肪细胞，形成大量脂滴，而正常对照组没有脂滴形成。给予PPARγ抑制剂处理后，MADD成纤维细胞脂滴明显减少。我们推测MADD疾病不仅存在线粒体功能障碍，还与脂肪生成异常有关，而PPARγ在脂质沉积过程中起着重要作用。为探索PPARγ在其发病机制中的作用，本课题拟建立MADD患者及正常人的iPS细胞，诱导分化为骨骼肌细胞，观察成肌诱导过程中脂滴的形成，进行线粒体功能、PPARγ及成脂相关基因等检测，观察PPARγ抑制剂处理后脂滴、线粒体等变化。本研究对MADD脂肪生成机制的研究将为临床治疗提供新的药物靶点。

多种酰基辅酶A脱氢酶缺乏症(MADD)是由ETF/ETFDH基因突变导致线粒体功能受损，引起脂肪酸β氧化障碍的遗传性代谢性肌病。目前发病机制尚不完全清楚。本研究采用MADD患者的皮肤成纤维细胞，研究MADD疾病骨骼肌肌纤维内脂滴异常增多的发病机制。主要进展如下：（1）建立了MADD患者iPS细胞，分化为骨骼肌细胞后，经成脂诱导后细胞内出现大量脂滴，而对照组没有脂滴。给予PPARγ抑制剂或维生素B2相关药物处理后，MADD细胞内脂滴明显减少。（2）采用CRISPR-Cas9基因编辑技术建立了Etfdh基因的纯合定点突变小鼠胚胎干细胞。Etfdh基因c.250G>A突变位点为中国患者，尤其是华南患者的最常见突变。同时观察到基因突变对于ES细胞多能性的维持和分化能力没有显著影响。（3）对ES细胞系进行体外诱导发现，定点突变ES细胞分化出的神经细胞、肝脏细胞、骨骼肌细胞等经成脂诱导中均出现明显的脂滴，提示该疾病的三个胚层的细胞均具有不同程度的成脂特性。目前临床中观察到患者的骨骼肌纤维中存在大量脂滴，影响患者的运动功能，同时部分患者在B超检查中发现不同程度的脂肪肝。本研究为探讨MADD疾病的发病机制奠定了坚实的工作基础，同时对于该疾病的临床认识提供了重要的启示性线索。