Wilson病基因产物在人肝细胞内存在和定位的研究

A preliminary study of presence and localization of the Wilson disease gene product in human hepatocytes.

目的 证实Wilson病铜转运P型ATP酶 (WD蛋白 )在人肝内的存在 ,并初步研究其在肝细胞内的具体定位。方法 应用Western blot蛋白印迹技术对离体培养正常人肝细胞内蛋白进行分离 ;通过高浓度铜及P型ATP酶阻滞剂矾酸钠、激动剂长春新碱孵育培养 ,差速离心分离肝细胞胞浆、溶酶体、线粒体和微粒体 ,对各亚细胞铜含量分别对照分析。结果 人肝细胞内分离出 1 5 5kDa的WD蛋白特异性条带 ;在人肝细胞质膜、微粒体内存在有铜转运P型ATP酶 ,其阻滞剂及激动剂对肝细胞铜转运有显著影响。结论 证实正常人肝细胞存在WD蛋白 ,其存在部分可能在肝细胞质膜、内质网和高尔基器。

Objective To confirm the presence and localization of Wilson disease gene product copper transporting P type ATPase (WD protein) in human hepatocytes. Methods Human cultured hepatocytes were incubated and the proteins were separated by SDS PAGE and analyzed by Western blot. Media of copper 15μg/mL only, copper 15μg/mL with vincristine (agonist of P type ATPase) 0.5μg/mL, or copper 15μg/mL with sodium vanadate (antagonist of P type ATPase) 18.39μg/mL of hepatocytes were used according to grouping. Microsomes, lysosomes, mitochondria, and cytosol of hepatocytes were isolated by differential centrifugation. The influence on copper transport of these organelles by sodium vanadate and vincristine was comparatively analyzed among different conditions. Results A specific 155 kDa band of WD protein was separated from normal human hepatocytes. Copper transporting P type ATPase was found to be localized in human hepatocytic plasma membranes and microsomes, and sodium vanadte and vincristine can obviously change their copper transporting. Conclusions WD protein plays an important role in hepatocytic copper metabolism. It was first directly confirmed that there were WD proteins in normal human liver, and this protein was probably situated at hepatocytic plasma membranes, Golgi apparatus and endoplasmic reticulum. Dysfunction of WD protein might be the most important pathogenetic factor of WD.