人ATP7B基因在转基因大鼠的铜代谢通路中的功能分析

Function Analysis of Human ATP7B in Copper Transport by Using Transgenic Rat

目的 利用转基因大鼠模型 ,研究人Wilson病致病基因ATP7B在铜代谢通路中的功能。方法 将构建的 7 1kb含有鸡 β肌动蛋白启动子的人正常ATP7BcDNA ,经显微注射法导入Wilson病动物模型LEC(long_evanscinnamon)大鼠受精卵的雄性原核。以RT_PCR和Westernblot分析其在转基因大鼠各组织内的表达谱 ;用抗大鼠铜蓝蛋白抗体检测肝细胞全铜蓝蛋白 (holoceruloplasmin)的合成 ;同时对 6～ 30周龄的转基因大鼠血清铜蓝蛋白亚铁氧化酶活性、血清铜浓度、胆汁的铜分泌量、以及肝、肾和脑组织中的铜含量进行分年龄段连续测定和统计学分析。结果 在转基因大鼠体内 ,ATP7B在各种组织中获得广泛表达 ,其中肝和肌肉中的表达水平较高 ,肝细胞恢复了全铜蓝蛋白的合成 ,血清中的铜含量及胆汁的铜排泄量增加 ,肝、肾组织中的铜蓄积减少。结论 完整表达人ATP7B基因产物的转基因大鼠的肝细胞 ,通过CPN介导的铜向血清的分泌和调节铜向胆汁的排泄 ,恢复了ATP7B参与铜转运功能 ,Wilson病的铜蓄积与ATP7B基因的突变直接相关。

Objective To investigate the functions of human ATP7B in copper metabolism \%in vivo\% by using the ATP7B transgenic rat. Method\ The 7 1 kb transgene consisting of human ATP7B cDNA under the control of chiken β_actin promoter was introduced into the LEC rats, an animal model of Wilson disease, by prenuclear microinjection. The expressions of human ATP7B transcript and protein in the tissues of the transgenic rats were detected by RT_PCR and Western blot. The biosythesis of holoceruloplasmin was determined by anti_rat ceruloplasmin antibody. The plasma ceruloplasmin ferroxidase activity, and the concentration of copper in plasma, bile, liver, kidney and brain were further examined at various intervals. Results\ The human ATP7B was universal expression in the tissues of transgenic rat, especially in liver and muscle. The biosynthesis of holoceruloplasmin was restored in hepatocyte of transgenic rats. The copper contents in plasma and in bile were significantly increased, while the dramatic decreases were noted both in the levels of hepatic and renal copper in the transgenic rats. Conclusion\ The intact ATP7B product compensated for the deficiency of the endogenous rattus protein and did function in intrahepatic copper transport by secreting copper into the plasma via incorporation into ceruloplasmin and by modulating the excretion of copper into the bile. The mutation of ATP7B is critical to the copper accumulations in Wilson disease.