Ⅱ型糖原累积病(Pompe病)骨骼肌脂质异常沉积的发生及致病机制研究

Pompe disease is an inherited disorder due to the deficiency of acid alpha-glucosidase (GAA) that results in glycogen accumulation in various tissues, with muscle weakness and cardiac hypertrophy being the main manifestations. The currently available enzyme replacement therapy (ERT) proved to be successful in reversing cardiac but not skeletal muscle abnormalities, indicating that glycogen accumulation is not the only causative factor. The massive lipid buildup was observed in Pompe disease patients and the GAA knockout C2C12 cell line that we have successfully established, but the mechanism and pathogenesis of lipid deposition in skeletal muscle are poorly understood. Our recent study has shown that the expression of Plin2, Plin3 and Plin5 were up-regulated and the expression of two key lipases ATGL and MGL were down-regulated, which resulted in the increase of lipid synthesis and the decrease of lipolysis in GAA knockout C2C12 myoblasts. Our previous study also found that the differentiation of GAA knockout C2C12 myoblasts into myotubes was attenuated, while decreasing lipid deposition via increased lipidolysis improved the differentiation. Thus, we put forward a viewpoint that ongoing muscle damage and dysfunction in Pompe disease may be explained by insufficient muscle differentiation and regeneration causing by the deposition of lipids. In this proposal, we will use GAA knockout C2C12 myoblasts to further observe the lipid metabolism and myogenic differentiation in Pompe disease. This study is expected to reveal the mechanism and pathogenesis of lipid deposition in skeletal muscle, and provide theoretical basis and potential targets for Pompe disease.

Pompe病是致残致死的隐性遗传病，患者主要表现为肌无力和心肌肥厚。GAA酶活性缺陷使糖原大量累积是直接致病因素，但酶替代治疗未能逆转肌无力的进展，提示糖原累积不是唯一病因。患者骨骼肌内可见脂质异常沉积，我们构建的GAA敲除C2C12细胞内脂质也显著增多，但脂质沉积的原因及其在骨骼肌病变中的作用尚无报道。预实验发现GAA敲除的C2C12细胞：脂滴包被蛋白Plin2、Plin3和Plin5表达上调引起脂质合成增多、脂肪分解酶ATGL和MGL表达下调导致脂质分解减少；向骨骼肌细胞分化障碍，而减少脂质沉积可改善其分化能力。由此，我们首次提出Pompe病骨骼肌脂质合成增多、分解减少使得脂质异常沉积，引起成肌分化障碍，进而影响骨骼肌的结构和功能。本研究将继续采用GAA敲除细胞模型，进一步观察脂质代谢和成肌分化过程的变化，深层次揭示脂质异常沉积发生的原因及其参与骨骼肌病变的机制，为疾病治疗提供新思路。

GAA酶活性缺陷使糖原大量贮积是Pompe病的直接致病因素。以往研究发现，除了糖原沉积外，患者骨骼肌内还有大量脂滴异常沉积。类似地，我们构建的GAA敲除C2C12细胞内脂滴数量增多、体积增大，但脂滴沉积的原因及其在骨骼肌病变中的作用尚不明确。本项目发现GAA基因敲除细胞内异常沉积的脂质主要是由磷脂包裹、富含甘油三酯的中性脂滴。深入研究发现甘油三酯合成关键酶的表达上调、脂滴生成重要结构蛋白脂滴包被蛋白的表达增加，以及甘油三酯水解关键酶表达减少是脂滴沉积的主要原因。本项目进一步研究发现，GAA敲除C2C12细胞线粒体内脂肪酸氧化关键酶表达下降使得游离脂肪酸氧化障碍、细胞能量利用不足，同时堆积的脂肪酸及其代谢物产生脂毒性，使得胞内活性氧水平升高、凋亡细胞比例增多。通过抑制甘油三酯合成关键酶DGAT1的表达，细胞内脂滴含量明显减少，然而减少细胞内脂滴的沉积并没有改善GAA敲除C2C12细胞的状态，胞内活性氧水平以及凋亡细胞比例反而更高。利用过表达载体增加DGAT1的表达可促进游离脂肪酸重新合成甘油三酯储存于脂滴，减少活性氧水平和凋亡细胞的比例。本项目初步揭示了游离脂肪酸重新合成甘油三酯储存于脂滴，可保护GAA敲除C2C12细胞免受活性氧化脂质毒性损伤的分子机制。本研究为脂代谢在Pompe病骨骼肌病变中的作用提供了新认识，为开展新的治疗方案提供理论依据。