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脂代谢相关基因在apoE基因缺失幼龄小鼠肝脏中的表达

叶红燕, 尹苗, 商允菊, 戴学栋, 张圣强, 荆文, 杜会芹, 张晾, 潘杰

山东师范大学生命科学学院,山东省动物抗性生物学重点实验室.山东,济南 250014

摘要

该文旨在分析脂代谢相关基因在载脂蛋白E（apolipoprotein E，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（wild type，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。alinE~(-/-)小鼠肝脏中apoAⅠ、almAⅣ表达在14d龄时即发生显著变化（{sl P}<0.05）；在1月龄时apoB100表达较同龄WT小鼠明显上调（{sl P}<0.05）；apoAⅤ表达则在2月龄时较同龄WT小鼠上调（{sl 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Angptl 3表达在3月龄时较同龄WT小鼠上调（{sl 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apoB 100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。

关键词： apoE基因缺失小鼠; 动脉粥样硬化; 脂代谢相关基因; 肝脏; RT-PCR

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

Ye Hongyan, Yin Miao, Shang Yunju, Dai Xuedong, Zhang Shengqiang, Jing Wen, Du Huiqin, Zhang Liang, Pan Jie

The Key Laboratory of Animal Resistant Biology of Shandong Province, College of Life Sciences, Shandong Normal University.Jinan 250014,Shandong

Abstract

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 #alpha# （PPAR#alpha#）, liver X receptor #alpha# （LXR#alpha#）, 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.

Key words: apoE knockout mice;Atherosclerosis;lipid metabolism related gene;Liver;RT-PCR

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引用本文：

叶红燕, 尹苗, 商允菊, 戴学栋, 张圣强, 荆文, 杜会芹, 张晾, 潘杰. 脂代谢相关基因在apoE基因缺失幼龄小鼠肝脏中的表达[J]. 生理学报 2008; 60 (1): 51-58.

Ye Hongyan, Yin Miao, Shang Yunju, Dai Xuedong, Zhang Shengqiang, Jing Wen, Du Huiqin, Zhang Liang, Pan Jie. Differential expressions of lipid metabolism related genes in the liver of young apoE knockout mice. Acta Physiol Sin 2008; 60 (1): 51-58 (in Chinese with English abstract).