ABCG5和ABCG8在肝细胞胆固醇转运中的作用

Role of ABCG5 and ABCG8 in cholesterol transport

目的验证ABCG5和ABCG8在胆固醇转运中的作用。方法通过脂质体介导将含ABCG5或ABCG5和ABCG8的质粒导入7721细胞系,通过抗生素G418、zeocin筛选获得新的细胞系7721-G5与7721-DG。用RT-PCR及Western blot等方法证明细胞系7721-G5与7721-DG中分别含有ABCG5,ABCG5和ABCG8。7721、7721-G5、7721-DG细胞系分别用[3H]标记的胆固醇、游离胆固醇、卵磷脂、无脂血清作用使细胞内胆固醇饱和,随后用牛磺胆酸钠/卵磷脂受体系统进行胆固醇外运实验。每60 min测定培养液中[3H]标记的胆固醇含量,计算外运的胆固醇数量。结果研究发现转染后含有ABCG5和ABCG8的7721-DG细胞系胆固醇转运能力明显高于其余两株细胞系,而仅含有ABCG5的7721-G5细胞系胆固醇转运能力无明显变化。结论ABCG5与ABCG8具有促进胆固醇外运的作用,两者必须形成异二聚体才能行使这种作用。此外,用myc和HA标记后并不影响ABCG5和ABCG8发挥功能。

Objective To determine the role of ABCG5 and ABCG8 in liver cholesterol transport, Methods The mouse ABCG5 cDNA alone or ABCG5 and ABCG8 cDNA together were transfected into 7721 cell line through lipofectamine 2000. New cell line 7721-G5 containing mouse ABCG5 cDNA and 7721-DG mouse confaining ABCG5 and ABCG8 cDNA was obtained through G418 and zeocin screening. The existence of ABCG5 and ABCG8 was proved by RT-PCR and Western blotting. The 7721, 7721-G5 and 7721-DG cells were all saturated with free cholesterol and radioactively labeled [^3H]-cholesterol. Cholesterol efflux began when the cells were cultured with taurocholate sodium/egg PC acceptors. The aliquots of medium were detected for [^3H]-cholesterol by liquid scintillation technique every 60 min. Results The 7721-EK3 cells showed increased cholesterol efflux than other two kinds of cells, while 7721-G5 cells had little change in cholesterol efflux. Conclusions ABCG5 and ABCG8 function as cholesterol transporters and promote cholesterol efflux under the circumstance that they 2 form the heterodimer. Meanwhile, ABCG5 or ABCG8 tagged with myc or HA can only slightly change its function.