wilson病基因产物在人肝细胞内存在和定位的研究

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1、Wilson病基因产物在人肝细胞内存在和定位的研究中国神经精神疾病杂志2000年第4期第26卷论著与学术交流作者：侯国庆　梁秀龄　陈嵘　黄帆　徐评议　欧翠华　王莹　唐莉雯单位：侯国庆　梁秀龄　陈嵘　黄帆　徐评议　欧翠华　王莹（中山医科大学附属第一医院神经科510080）；唐莉雯（DepartmentofPharmacology,UniversityofKentucky,KY40506）关键词：肝豆状核变性；P型ATP酶；铜；肝细胞　　【摘要】　目的　证实Wilson病铜转运P型ATP酶(WD蛋白)在

2、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155kDa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。

3、ApreliminarystudyofpresenceandlocalizationoftheWilsondiseasegeneproductinhumanhepatocytesHuangFan　HouGuoqing　LiangXiuling　ChenRong　LiwenTang　HuangFan　XuPingyi　OuCuihua　WangYing　　（DepartmentofNeurology,TheFirstAffiliatedHospital,SunYat-senUniversityofMe

4、dicalSciences,Guangzhou.510080.Tel:020-87755766-8282）　　【Abstract】Objective　ToconfirmthepresenceandlocalizationofWilsondiseasegeneproduct-coppertransportingP-typeATPase(WDprotein)inhumanhepatocytes.MethodsHumanculturedhepatocyteswereincubatedandtheprote

5、inswereseparatedbySDS-PAGEandanalyzedbyWestern-blot.Mediaofcopper15μg/mLonly,copper15μg/mLwithvincristine(agonistofP-typeATPase)0.5μg/mL,orcopper15μg/mLwithsodiumvanadate(antagonistofP-typeATPase)18.39μg/mLofhepatocyteswereusedaccordingtogrouping.Micro

6、somes,lysosomes,mitochondria,andcytosolofhepatocyteswereisolatedbydifferentialcentrifugation.Theinfluenceoncoppertransportoftheseorganellesbysodiumvanadateandvincristinewascomparativelyanalyzedamongdifferentconditions.Results　Aspecific155kDabandofWDpro

7、teinwasseparatedfromnormalhumanhepatocytes.CoppertransportingP-typeATPasewasfoundtobelocalizedinhumanhepatocyticplasmamembranesandmicrosomes,andsodiumvanadteandvincristinecanobviouslychangetheircoppertransporting.Conclusions　WDproteinplaysanimportantro

8、leinhepatocyticcoppermetabolism.ItwasfirstdirectlyconfirmedthattherewereWDproteinsinnormalhumanliver,andthisproteinwasprobablysituatedathepatocyticplasmamembranes,Golgiapparatusandendoplasmicreticulum.DysfunctionofWDproteinmightbethemos