磷酸二酯酶Ⅲ抑制剂治疗Ⅰ型糖原累积病的实验研究

Mutation in G6PC is the main cause of a disable and mortal disease named glycogen storage disease typeⅠ. It makes glucose-6-phosphatase catalytic unit fail to hydrolyze glucose-6-phosphate for enzymatic defect. Reduplicative hypoglycemia, hepatomegaly, and liver dysfunction are clinical characteristics of GSD type Ⅰ. No efficient treatment to control the disease and it makes patients worse life. According to our clinical finding, mutation in G6pcdecreased mRNA expression of G6pc. Stimulation of glucagon could level up patients' blood glucose if they were mild phenotype. It is known that enzymatic reaction mediated by cAMP is the molecular mechanism of glucagon to level up blood glucose. As the concentration of cAMP would be decreased with phosphodiesterase hydrolysis, a hypothesis is drawn out that when the hydrolyze of cAMP was inhibited by phosphodiesterase Ⅲ inhibitors, the affect of cAMP would be prolonged to up-regulate G6PC mRNA expression and increaseG6PC protein transcription, which would be a therapy to glycogen storage disease typeⅠ. Comparable drug intervention by PDE3 inhibitors and glucagon to G6PC mutation knock-in mouse model would be processed in vivo and in vitro to verify the correction of the hypothesis. This study will form an experimental foundation for the precise treatment of glycogen storage disease type Ⅰwith PDE3 inhibitor.

Ⅰ型糖原累积病（GSDⅠ）是因G6PC基因突变致葡萄糖-6-磷酸酶（G6Pase）酶效力下降、糖异生和糖原分解障碍的遗传病，临床表现为低血糖、肝肿大、肝硬化，目前无有效药物治疗。本项目前期研究发现①GSDⅠ患儿G6Pase mRNA表达下调；②胰高血糖素刺激可使部分患儿血糖升高。研究证实胰高血糖素是通过cAMP酶促反应失活糖原合成酶，抑制糖原合成，并上调G6Pase mRNA表达促进糖原分解，使血糖上升。据此申请人提出科学假说：以磷酸二酯酶Ⅲ（PDE3）为靶点，运用PDE3抑制剂抑制cAMP水解，提高cAMP浓度，上调G6Pase mRNA表达，可以增加G6Pase蛋白转录，治疗GSDⅠ。并拟构建G6PC点突变敲入小鼠，应用胰高血糖素与PDE3抑制剂治疗，观察给药前后野生型与突变型小鼠生长发育、肝脏结构、基因表达、酶活性、糖原合成分解变化，评价PDE3抑制剂药效，为治疗GSDⅠ提供新策略。

Ⅰ型糖原累积病被列入《第一批罕见病目录》，其诊断与治疗受到罕见病研究团队的关注。Ⅰ型糖原累积病是一种严重致残致死的单基因遗传病，发病率约为十万分之一，可分为由G6PC基因突变引起的Ⅰa型糖原累积病与SLC37A4基因突变引起的Ⅰb型糖原累积病，Ⅰa型糖原累积病患儿临床特征包括生长发育迟缓、肝大、低血糖、高乳酸、高脂血症等，Ⅰb型糖原累积病患儿除了有上述临床特点外，兼有中性粒细胞减少、中性粒细胞功能缺陷等免疫缺陷，出现反复感染，危害生命。Ⅰ型糖原累积病目前尚无有效药物治疗，只能通过食用生玉米淀粉控制病情进展，这为患者家庭与社会带来沉重负担。本课题的开展立足于前期临床基础，通过构建动物疾病模型研究已有药物治疗Ⅰ型糖原累积病的可能性。本课题获得成果包括：①成功构建了G6PC基因敲除小鼠的Ⅰa型糖原累积病小鼠模型；②成功构建携带中国地区SLC37A4基因热突变G149E的Ⅰb型糖原累积病小鼠模型；③通过背景优化的技术获得了SLC37A4 G149E/G149E纯合子，SLC37A4 G149E/G149E纯合子能成功模拟Ⅰb型糖原累积病生长发育迟缓、肝大、低血糖、高乳酸、高脂血症、中性粒细胞减少等临床症状，为探索Ⅰb型糖原累积病发病机制提供了便利的实验工具；④成功分离得到模型小鼠成纤维细胞，为后续构建筛选与开发治疗Ⅰb型糖原累积病的细胞平台奠定基础；⑤利用成功构建的动物模型和细胞模型，探索了PDE抑制剂、GLUT2抑制剂对Ⅰb型糖原累积病小鼠的影响，实验结果提示GLUT2抑制剂有助于提高幼鼠生存率。本项目的研究结果，建立了Ⅰ型糖原累积病药物筛选的细胞与动物模型，并对PDE抑制剂罗氟司特、GLUT2抑制剂恩格列净治疗Ⅰ型糖原累积病进行实验探索，为后续Ⅰ型糖原累积病治疗药物研究提供了实验工具与研究方向。