脂代谢相关基因在apoE基因缺失幼龄小鼠肝脏中的表达

Differential expressions of lipid metabolism related genes in the liver of young apoE knockout mice

本文旨在分析脂代谢相关基因在载脂蛋白E(apolipoproteinE,apoE)基因缺失(apoE-/-)幼龄小鼠肝脏中的表达特征及其与血脂紊乱和动脉粥样硬化(atherosclerosis,AS)早期病变的关系。利用半定量RT-PCR和荧光实时定量RT-PCR技术,分析14d龄、1、2和3月龄apoE-/-小鼠及同龄野生型(wildtype,WT)小鼠肝脏脂代谢相关基因的表达,并进行血生化指标及主动脉病理形态学检测。apoE-/-小鼠肝脏中apoAI、apoAIV表达在14d龄时即发生显著变化(P<0.05);在1月龄时apoB100表达较同龄WT小鼠明显上调(P<0.05);apoAV表达则在2月龄时较同龄WT小鼠上调(P<0.05),此时可观察到apoE-/-小鼠主动脉内膜出现AS早期病变;Fat/CD36和Angptl3表达在3月龄时较同龄WT小鼠上调(P<0.05);实验中检测的其它基因的mRNA表达与同龄WT小鼠相比无显著性差异。apoE-/-小鼠血清总胆固醇、甘油三酯、低密度脂蛋白胆固醇和高密度脂蛋白胆固醇含量均高于同龄WT小鼠,并随年龄增长而升高。apoE-/-小鼠和同龄WT小鼠血清中apoB100蛋白浓度在14d龄到3月龄间变化趋势与其在肝脏中mRNA表达及血清中低密度脂蛋白胆固醇含量变化趋势基本一致。上述部分脂代谢相关基因表达在幼龄小鼠即发生改变,与血脂紊乱以及主动脉AS病变发生发展过程呈正相关,说明其可能在幼龄小鼠脂质代谢紊乱发生过程中起重要作用,从而引起动脉内皮细胞功能改变乃至AS早期病变的发生。

The work was aimed to investigate the differential expressions of lipid metabolism related genes in the early stage of atherosclerosis in the young apolipoprotein E deficient （apoE^-/-） mice at different ages with normal chow diet. The genotypes of mice were identified by using multiplex polymerase chain reaction （multi-PCR） analysis. The semi-quantitative reverse transcriptionpolymerase chain reaction （RT-PCR） and real-time quantitative RT-PCR were used to analyze the expressions of lipid metabolism related genes in the liver of apoE4 and age-matched wild type （WT） mice of 14-day old, 1-month old, 2-month old, 3,month old. The serum total cholesterol （TC）, triglycedride （TG）, low-density lipoprotein cholesterol （LDL-C） and high-density lipoprotein cholesterol （HDL-C） contents were assayed using COD-PAP and GPO-PAP methods. The serum apolipoprotein B100 （apoB 100） content was quantitated by immune turbidimetry. The hearts were perfusion-fixed in 4% formaldehyde, infiltrated with 30% gum sucrose for 24 h at 4℃, and embedded in OCT compound. The aortic sinus tissues were serially sectioned at -15℃, stained with Sudan Ⅳ, and counterstained with light green. The results were shown as follows. Compared with that in WT mice, the mRNA levels of apoA Ⅰ and apoA Ⅳ in apoE^-/- mice aged from 14-day old to 3-month old changed prominently （P〈0.05）, with apoA Ⅰ up-regulated and apoA Ⅳ down-regulated. At the age of 1 month, the expression of apoB 100 in apoE/ mice was higher than that in WT mice （P〈0.05）. The expression of apoA Ⅴ was up-regulaied （P〈0.05） and there was obvious lipid deposition in the aortic intima in apoE^-/- mice at the age of 2 months. The expressions of fatty acid translocasc （Fat/CD36） and angiopoietin-like protein 3 （Angptl 3） in apoE^-/- mice were higher than those in WT mice at the age of 3 months （P〈0.05）, while the expressions of peroxisome proliferator-activated receptor α （PPARα）, liver X receptor α （LXRα）, carnitine palmitoyl transferase Ⅰ （CPT Ⅰ） and acyl coenzyme A oxidase Ⅰ （ACOXI） showed no significant changes. The serum TC, TG, LDL-C and HDL-C contents in apoE^-/- mice aged from 14-day old to 3-month old were higher than those in age-matched WT mice. apoE^-/- mice showed a marked increase in serum apoB100 content, consistent with the trend of serum LDL-C content and apoB100 mRNA content in the liver. The results suggest that the mRNA expressions of apoA Ⅰ, apoA Ⅳ, apoA Ⅴ, apoB 100 and Angptl 3 in apoE^-/- mice change significantly compared with those in WT mice, and these genes might be relevant to the complicated lipid metabolism network, and involved in the early stage of atherogenesis.