Functional expression of Wilson's disease gene in the LEC rats by adenovirus-mediated gene delivery.

WND基因（正式命名为ATP 7 B）编码一种假定的铜转运P型ATP酶，在Wilson病患者中是缺陷的，导致铜在肝脏中过度积聚。Long-Evans Cinnamon（LEC）大鼠是Wilson病的啮齿动物模型，其临床表现与Wilson病相似。Atp 7 b是WND的大鼠基因同源物，在大鼠中也有缺陷。为了研究WND蛋白在体内的功能及其在细胞内的定位，本研究利用重组腺病毒介导的基因转移技术将WND cDNA导入LEC大鼠体内。将含有WND cDNA的重组腺病毒（1 × 10 ~ 4噬<10>斑形成单位）尾静脉注射给4-6周龄LEC大鼠。免疫荧光和亚细胞分级分析表明，转基因在肝脏中表达，并定位于高尔基体。此外，由于LEC大鼠中全血浆铜蓝蛋白的合成受到干扰，因此检查了全血浆铜蓝蛋白、氧化酶活性和铜结合形式的血浆水平，以评价WND蛋白在铜转运方面的功能。因此，通过Western印迹分析和血浆中氧化酶活性和铜含量的测量证实血浆中全铜蓝蛋白的出现。结论：1）WND蛋白可能参与铜蓝蛋白的合成和铜转运。2)高尔基体可能是WND蛋白发挥功能的部位。3)腺病毒介导的基因传递可能是治疗Wilson病的一种可能方法。

WND gene (officially designated ATP7B), which encodes a putative copper transporting P-type ATPase, is defective in the patients with Wilson's disease, resulting in the excessive accumulation of copper in the liver. The Long-Evans Cinnamon (LEC) rat, known as a rodent model for Wilson's disease, shows some of clinical features similar to Wilson's disease. Atp7b, the rat gene homologue to WND,is also defective in the rat. To investigate the in vivo function of WND protein as well as its intracellular localization, WND cDNA was introduced to the LEC rats by recombinant adenovirus mediated gene delivery. The recombinant adenoviruses containing WND cDNA,1X10^<10> plaque forming units, were administered to 4-6 week old LEC rats by tail vein injection. Immunofluorescent study and subcellular fractionation study revealed the transgene expression in liver and its localization to the Golgi apparatus. Moreover, since the synthesis of holoceruloplasmin is disturbed in the LEC rat, the plasma level of holoceruloplasmin, oxidase active and copper bound form, was examined to evaluate the function of WND protein with respect to the copper transport. Consequently, the appearance of holoceruloplasmin in plasma was confirmed by Western blot analysis and plasma measurements for the oxidase activity and the copper content. Conclusions : 1) Introduced WND protein may function in the copper transport coupled with the synthesis of ceruloplasmin. 2) The Golgi apparatus is the likely site for WND protein to manifest its function. 3) Adenovirus mediated gene delivery could be a possible treatment for Wilson's disease.