Dicer1 敲除对肝脏胆酸代谢和转运功能的影响

目的:微小RNA(microRNAs,miRNAs)在胆固醇的合成,代谢和转运中起着重要作用,而mi RNAs在胆固醇代谢物胆酸的代谢和转运中的作用尚不清楚。Dicer基因是miRNAs生成过程的关键酶。本课题使用肝脏特异的Dicer1基因敲除小鼠,考察肝脏Dicer1基因敲除对C57BL/6小鼠肝脏胆酸代谢和转运的影响。方法:使用白蛋白启动子驱动的Cre重组酶和Loxp系统(Alb-Cre/Loxp)在小鼠肝脏中特异的敲除Dicer1基因;分别收集3~12周龄的小鼠血液和肝脏组织,使用Cobas生化仪检测小鼠血液和肝脏中总胆酸含量;利用实时定量PCR的方法分析肝脏中胆汁酸代谢转运相关基因的表达。结果:实验发现,肝脏Dicer基因敲除后,胆酸在血液和肝脏中明显蓄积,弥漫性肝细胞轻微空泡化,偶见单个肝细胞坏死。检测胆酸代谢和转运相关基因的表达发现,胆酸合成相关基因的表达有轻度升高,但缺乏统计学差异;在肝脏细胞血管侧的胆酸摄取转运体中,Oatp1a1在Dicer1敲除小鼠肝脏中明显下调,Ntcp和Oatp1b2则无明显改变;而肝细胞血管侧胆酸外排转运体的表达均有显著升高,胆管侧的外排转运体中Abcb11表达有明显增加。结论:Dicer基因敲除后,胆酸在血液和肝脏中明显蓄积,肝脏和血液中胆酸总量显著增加。血液中胆酸的蓄积可能与肝脏细胞血管侧摄取转运体的低表达和血管侧外排转运体的高表达有关;而肝脏中胆酸的蓄积可能部分来自于轻度升高的胆酸合成酶,胆酸在肝细胞内运输途径的紊乱可能与肝脏和血液中胆酸总量的显著增加相关。

Effect of Dicer1-deficient Liver on Metabolismand Transport Function of Bile Acid

Objective:MicroRNA (miRNA) plays a pivotal role in cholesterol synthesis, metabolismand transport, but the role of miRNAs in bile acid metabolism and transport is unclear. Dicer is the key enzyme of miRNA biogenesis. This study focused on the impact of Dicer1 knockout on bile acid metabolismand transport in C57BL/6 mice.Methods:Cre recombinase driven by Albumin promoter and the floxed dicer allele were used for Dicer1 knockout in the liver of mice. Cobas biochemical analyzer was used to test bile acid level in the blood and liver; Real-time quantitative PCR was used to analyze gene expression.Results:In Dicer1 knockout mice, bile acids accumulated in the blood and liver, diffuse vacuolation of hepatocytes was observed, the single cell necrosis was found occasionally. Bile acid synthesis gene expression was slightly increased but without statistical difference; Oatplal, a bile acid uptake transporter, was obviously decreased in the dicer1 knockout mice. No obvious change can be seen in the expression of Ntcp and Oatp1b2; however, bile acid efflux transporters at the basolateral membrane were markedly increased, and Abcb11, the major efflux transporter of bile acid at canalicular membrane, did not show obvious change.Conclusion:Bile acid accumulated in the blood and liver of dicer1 knockout mice. The bile acid accumulation in blood may be related to low expression of uptake transporter and high expression of efflux transporter at the basolateral membrane. The bile acid accumulation in liver may be related to slightly increased bile acid synthesis enzyme. The disorder of bile acid trafficking may be related to increased total bile acid in the liver and blood.