Production and analyses of diverse models for human Wison's disease

LEC大鼠，威尔逊病的动物模型，有一个与Atp7b同源的Atp7b基因缺失，表现出一些与威尔逊病相似的临床特征。为了进一步确定ATP7B基因在体内铜代谢中的功能及其与肝脏疾病的关系，构建了带CXN启动子的人ATP7B cDNA，并通过核前微注射引入LEC大鼠。转基因大鼠（Tg）在肝、肾、脑、肌肉等多个组织中表达人ATP7B转录本和蛋白。证实Tg血浆中铜蓝蛋白氧化铁酶活性和铜浓度升高，与western blot检测到的全息蓝蛋白（Holo-CPN）的出现相对应。从16周龄开始，Tg胎鼠胆汁中的铜含量比非Tg胎鼠增加了近2倍。甘油三酯组的GOT和GPT活性明显低于非甘油三酯组。Tg组大鼠的长期存活率约为97%，而非Tg组的存活率为26.3%。Tg大鼠的长期团队存活率约为97%，而非Tg的幼崽存活率为26.3%。这些结果和组织学检查表明，Tg大鼠从暴发性肝炎中获救，并表现出晚发性肝纤维化。此外，ATP7B衍生的产物通过与CPN结合和通过胆道排泄途径参与细胞间铜运输。这些结果表明，人ATP7B可以通过基因转移在体内部分补充大鼠ATP7B的功能。在Tg肝中也发现了明显的肝铁剥夺，提示ATP7B也可能在铁代谢中起作用，铁可能在Wilson病肝脏异常的发展中起重要作用。此次成功拯救Tg大鼠对肝豆状核变性的基因治疗具有重要意义，Tg系统将有助于进一步研究ATP7B的功能、其与铜或铁代谢的关系以及肝脏疾病的发展。少

The LEC rat, an animal model of Wilson's disease, has a deletion in Atp7b gene homologous to the ATP7B and shows some of the clinical features similar to Wilson's disease. To further ascertain the function of ATP7B gene in copper metabolism and its relation to hepatic disease in vivo, human ATP7B cDNA with CXN promoter was constructed and introduced into the LEC rats by prenuclear microinjection. The transgenic rats (Tg) expressed human ATP7B transcripts and protein in their multiple tissues, including liver, kidney, brain and muscle etc. The elevated ceruloplasmin ferroxidase activity and copper concentration in Tg plasma was confirmed, corresponding to the appearance of holoceruloplasmin (Holo-CPN) detected by western blot analysis. Copper content in Tg bile increased by nearly 2 fold of that in non-Tg littermates from 16 weeks of age. GOT and GPT activities in Tg much decreased compared with those in non-TG littermate. Long-term survival in Tg rats was up approximately to 97%, in co … More ntrast with 26.3% survival in non-Tg littermates. Long-team survival in Tg rats was up approximately to 97%, in contrast with 26.3% survival in non-Tg littermates. These findings and histological examination indicate that Tg rats were rescued from fulminant hepatitis and exhibited late onset of hepatic fibrosis. In addition, the products derived from ATP7B involve in intercellular copper transport by incorporation with CPN and by biliary excretion pathway. These results indicated that human ATP7B could partially complement the function of rat atp7b by gene transfer in vivo. Significant deprivation of hepatic iron from the Tg liver was also found, indicating that ATP7B may also act function in iron metabolism, and iron probably plays an essential role in the development of hepatic abnormalities in Wilson's disease. This successful rescue of Tg rats has important implication for the gene therapy for Wilson's disease and the Tg system will be useful to further study the function of ATP7B, its relation to copper or iron metabolism and the development of hepatic disease. Less