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

WND基因(正式命名为ATP7B)编码一种可能的铜转运P型ATPase，在肝豆状核变性患者中存在缺陷，导致铜在肝脏中过度蓄积。Long-Evans Cinnamon(LEC)大鼠是威尔逊病的啮齿动物模型，表现出一些类似威尔逊病的临床特征。与WND同源的大鼠基因ATP7B在大鼠身上也存在缺陷。为研究WND蛋白在体内的功能及其在细胞内的定位，以重组腺病毒为载体将WND基因导入LEC大鼠体内。4~6周龄LEC大鼠尾静脉注射含WND基因的重组腺病毒1X10；免疫荧光研究和亚细胞分离研究表明转基因基因在肝脏中表达，并定位于高尔基体。此外，由于LEC大鼠全血铜蓝蛋白的合成受到干扰，因此我们检测了WND蛋白在铜转运过程中的血浆水平，以评价WND蛋白在铜转运中的作用。因此，通过蛋白质印迹分析和血浆中氧化物酶活性和铜含量的测定，证实了血浆中存在全角蓝蛋白。结论：1)导入的WND蛋白可能参与铜转运和铜蓝蛋白的合成。2)高尔基体可能是WND蛋白发挥功能的部位。3)腺病毒介导的基因治疗可能成为治疗肝豆状核变性的一种方法。

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.