靶向激活AMPK抑制糖原合成酶对糖原累积病Ⅸa型糖原沉积的治疗作用及机制研究

Glycogen storage disease type Ⅸa (GSDⅨa) is a rare X-linked recessive genetic disease caused by mutations in the PHKA2 gene, which encodes glycogen phosphorylase kinase (PhK). By catalyzing inactive phosphorylase into active form to initiate glycogen degradation, PhK plays an important role in regulating metabolism of glycogen. Mutations in the PHKA2 gene cause deficiency of PhK in the cells, leading to accumulative deposition of glycogen in the liver. GSDⅨa is characterized by hepatomegaly, elevated transaminase and chronic hepatic fibrosis which has no effective therapy. In our previous clinical study we found that the expression level of glycogen phosphorylase was decreased in GSDⅨa patients, but that of glycogen synthase was increased, which deteriorated the glycogen acumination in the liver. With study of PHKA2 knock-down HepG2 cells by siRNA interference, we observed the same changes of expression in glycogen phosphorylase and glycogen synthase. To explore an effective treatment of GSDⅨa, we further investigated the potential effect and mechanism of berberine, a known AMPK activator, and observed the decrease in the expression of glycogen synthase and the reduction of glycogen acumination in the PHKA2 knock-down HepG2 cells after treated with berberine. We hypothesize that intensive glycogen synthesis aggravates the accumulative deposition of glycogen in the liver, and while through activating AMPK, the increased glycogen synthase in the liver of GSDIXa could be inhibited and the reduction of glycogen acumination could be achieved. In order to investigate the therapeutic effect and mechanism of AMPK activation on the inhibition of glycogen synthesis, we will first construct PHKA2 knock-out L02 liver cell line, and investigate the accumulative deposition of glycogen, the expression of glycogen phosphorylase, glycogen synthase and their enzyme activities. These parameters will be compared and analyzed before and after AMPK activation and inhibition respectively. We also plan to create PHKA2 gene knock-out mouse model, test the phenotype including height, weight, liver volume, and biochemical and molecular characterizations as well. Before and after AMPK activation and inhibition respectively, the differences of the expression level of glycogen phosphorylase, and glycogen synthase and their enzyme activities in the liver will be investigated and compared between PHKA2 gene knock-out mice and controls. The aims of this project are to study the pathogenic role of increased glycogen synthesis in GSDⅨa, reveal the therapeutic effect of AMPK activator on glycogen acumination, and probe into the molecular mechanism of AMPK activation, which might provide us a potential new therapy for GSDⅨa.

糖原累积病Ⅸa（GSDⅨa）是罕见遗传病，因PHKA2基因突变致磷酸化酶激酶功能缺陷，糖原不能分解沉积于肝，致肝大、纤维化，无有效治疗药物。我们前期临床发现该病不仅糖原分解障碍，同时糖原合成酶表达亢进，加剧糖原沉积；随后在PHKA2敲低HepG2细胞中证实了此现象，并发现AMPK激动剂黄连素可抑制糖原合成酶表达、减少糖原沉积。据此首次提出糖原合成亢进参与GSDIXa致病，靶向激活AMPK抑制糖原合成酶可治疗GSDIXa糖原沉积。本项目拟构建PHKA2敲除L02肝细胞系，检测糖原沉积、糖原磷酸化酶及合成酶表达和活性；同时构建PHKA2敲除小鼠模型，观察小鼠身长体重、肝体积和肝糖原沉积、糖原磷酸化酶及合成酶表达和活性；并激活和抑制AMPK，观察细胞和小鼠模型上述指标变化，最终揭示糖原合成亢进参与GSDⅨa肝糖原沉积新分子机制和靶向激活AMPK抑制肝糖原合成酶从而治疗GSDⅨa肝糖原沉积新方法。

本项目拟构建PHKA2敲除L02肝细胞系，检测糖原沉积、糖原磷酸化酶及合成酶表达和活性；同时构建PHKA2敲除小鼠模型，观察小鼠身长体重、肝体积和肝糖原沉积、糖原磷酸化酶及合成酶表达和活性；并激活和抑制AMPK，观察细胞和小鼠模型上述指标变化，揭示糖原合成亢进参与GSDⅨa肝糖原沉积新分子机制和靶向激活AMPK抑制肝糖原合成酶从而治疗GSDⅨa肝糖原沉积新方法。.课题组按计划构建了PHKA2敲除L02肝细胞系并检测糖原沉积、糖原磷酸化酶及合成酶表达和活性；构建了PHKA2基因敲除小鼠模型与PHKA2基因点突变敲入小鼠模型，观察小鼠身长体重、肝体积和肝糖原沉积、糖原磷酸化酶及合成酶表达和活性，并通过改变饲养条件，诱发疾病表型。但由于细胞模型与小鼠模型均未体现明显的病理特征，未能完全模拟GSDⅨa患者代谢情况，因此未能使用AMPK激活剂与抑制剂对肝糖原的沉积机制进行探究。.为建立更为可靠的研究模型，课题组构建了GSDⅨa患者外周血单核细胞来源诱导干细胞iPSC-GSDⅨa，定向诱导分化为类肝细胞并进行验证。GSDⅨa患者来源的诱导多能干细胞系GSDⅨa-iPSC在体外能稳定传代，细胞核型长期保持稳定；多能干细胞标志蛋白Tra-1-60、SSEA4、Oct4、Nanog表达阳性，能够保持细胞多能性；在体外能形成EBs，EBs分化后三胚层多能性标志蛋白Nestin、α-SMA、SOX17表达阳性，证明细胞系拥有多胚层分化的能力。课题组成功实现GSDⅨa-iPSCs向HLCs的定向分化，能合成肝细胞特异性蛋白AFP、ALB；能实现肝细胞的特异性功能如吸收LDL、吸收与排泄吲哚氰绿和生成糖原；在疾病表型方面，GSDⅨa-HLCs的PHKA2基因mRNA表达显著下降，PHKA2蛋白不表达，有更多糖原累积（P<0.05）。GSDⅨa患者来源的诱导多能干细胞系GSDⅨa-iPSC的建立，可以为后续研究提供充足的细胞来源。定向分化获得的GSDⅨa-HLCs 能够模拟GSDⅨa患者的肝脏病理特征，可以被认为是可靠的GSDⅨa病理模型，为后续致病机制和治疗研究奠定了良好基础。后续研究将继续通过该类肝细胞模型探索PHKA2基因缺陷所致的糖原累积病Ⅸa发病机制与治疗方案。