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apoE/LDLR双基因缺失幼龄小鼠主动脉中动脉粥样硬化相关基因的表达

戴学栋, 尹苗, 荆文, 杜会芹, 叶红燕, 商允菊, 张晾, 邹艳艳, 曲志萍, 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；济南市中心医院医学实验室诊断中心.山东,济南 250013

摘要

利用RT--PCR以及实时定量RT--PCR检测11个动脉粥样硬化（atherosclerosis，AS）相关基因在1、2和3月龄的载脂蛋白E（apolipoprotein E，aopE）／低密度脂蛋白受体（low--density lipoprotein receptor，LDLR）双基缺失（apoE~(-/-)／LDLR~(-/-)）小鼠主动脉中的表达变化，同时应用血生化指标和病理形态学观察AS早期病变特点，探讨apoE和LDLR基因联合缺失引发的血脂代谢紊乱和血管炎症损伤的关系以及AS的炎症反应机制。结果显示，apoE~(-/-)／LDLR~(-/-)小鼠IL--1#beta#、TLR2、MCP--1、ICAM--1、VCAM--1、GM--CSF、CD36和ET--1表达在1月龄时较同龄野生型（wild type，WT）小鼠显著上调（{sl P}<0.05，{sl P}<0.01），PDGF--#alpha#和TNF--#alpha#表达在2月龄时较同龄WT小鼠显著上调，除ET--1表达在2月龄时以及TLR2、VCAM--1和ICAM--1表达在3月龄时降至WT小鼠水平以外，其余各基因表达随年龄增长继续升高（{sl P}<0.05，{sl P}<0.01），其中MCP--1表达在2月龄时达到峰值。NF--#kappa#B在各年龄段apoE~(-/-)／LDLR~(-/-)小鼠中的表达与同龄WT小鼠相比均无显著差异。各年龄段apoE~(-/-)／LDLR~(-/-)小鼠血清TC、TG、LDL、HDL、TNF--#alpha#、IL--1#beta#和ox--LDL含量均显著高于同龄WT小鼠（{sl P}<0.05，{sl P}<0.01），并随年龄增长逐渐升高。apoE~(-/-)/LDLR~(-/-_小鼠1月龄时主动脉内膜出现少量的散在的脂质沉积，随着年龄增长病变区域增多，脂质沉积增厚。上述结果提示：apoE和LDLR双基因缺失形成的高脂血症可能通过刺激主动脉中炎症基因时序表达，起始并扩大病变部位的炎症反应，共同促进AS的发生发展。

关键词： apoE~(-/-)/LDLR~(-/-)小鼠; 动脉粥样硬化; 主动脉; RT-PCR; 动脉粥样硬化相关基因

Expressions of atherosclerosis--related genes in aorta in young apoE/LDLR double knockout mice

Dai Xuedong, Yin Miao, Jing Wen, Du Huiqin, Ye Hongyan, Shang Yunju, Zhang Liang, Zou Yanyan, Qu Zhiping, Pan Jie

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

Abstract

To systematically clarify the effects of apolipoprotein E （aopE） and low-density lipoprotein receptor （LDLR） gene mutant on hyperlipidemia, vascular inflammation impairment and pathogenesis of atherosclerosis （AS）, total RNA was isolated from fresh aortas of young apoE/LDLR double knockout （apoE~(-/-)/LDLR~(-/-)） and wild type （WT） mice using TRIzol reagent. Then RNA was reversely transcribed to first-strand cDNA by reverse transcriptase for reverse transcription polymerase chain reaction （RT-PCR） and real-time RT-PCR. Primer pairs were designed using primer design software according to the gene sequences available in GenBank. #beta#-actin was used as an internal control. Then RT-PCR assay was used to analyze the expression patterns of interleukin-1#beta# （IL-1#beta#）, minor necrosis factor-#alpha# （TNF-#alpha#）, nuclear factor-#kappa#B （NF-#kappa#B）, colony-stimulating factor （GM-CSF）, CD36, endothelin-1 （ET-1）, toll-like receptor 2 （TLR2）, monocyte chemoattractant protein-1 （MCP-1）, vascular adhesion molecule-1 （VCAM-1）, intercellular adhesion molecule-1 （ICAM-1） and platelet-derived growth factor-#alpha# （PDGF-#alpha#）. SYBR Green quantitative real-time RT-PCR was used to validate gene expressions identified by RT-PCR. Blood samples were taken from the retro-orbital venous plexus, and serum levels of total cholesterol （TC）, triglyceride （TG）, low-density lipoprotein （LDL） and high-density lipoprotein （HDL） were measured by using biochemical techniques. Serum concentrations of circulating TNF-#alpha#, IL-1#beta# and oxidized LDL （ox-LDL） were determined by ELISA. Frozen sections of aortic sinus were stained with Sudan IV to visualize intimal fatty lesions. The results showed that the relative expressions of IL-1#beta#, GM-CSF, ET-1, TLR2, CD36, MCP-1, ICAM-1 and VCAM-1 in apoE~(-/-)/LDLR~(-/-) mice at the age of 1 month were higher than those in age-matched WT mice （P〈0.05, P〈0.01）, respectively. The expressions of PDGF-#alpha# and TNF-#alpha# in apoE~(-/-)/LDLR~(-/-) mice at the age of 2 months were up-regulated compared to those in age-matched WT mice （P〈0.05）. All the expressions of target genes continued to be up-regulated （P〈0.05, P〈0.01） except that ET-1 expression at the age of 2 months, TLR2, VCAM-1 and ICAM-1 expressions at the age of 3 months were down-regulated to that in WT mice. NF-#kappa#B expression had no significant changes between two genotype mice at different ages. All the gene expressions kept unchanged in WT mice at different ages, except that IL-1#beta# expressions were slightly up-regulated at the ages of 2 and 3 months. Serum levels of TC, TG, LDL, HDL, TNF-#alpha# IL-1#beta# and ox-LDL in apoE~(-/-)/ LDLR~(-/-) mice at different ages were higher than those in age-matched WT mice （P〈0.05, P〈0.01）, and were increasing with age. Primary atherosclerotic lesions were observed in 1-month old apoE~(-/-)/LDLR~(-/-) mice and were progressing with age. There were no lesions observed in all WT mice at different ages. The data suggest that hyperlipidemia due to apoE and LDLR gene mutant may stimulate the temporal expressions of AS-related genes and contribute to primary atherogenetic lesions and vascular inflammation impairment.

Key words: apoE~(-/-)/LDLR~(-/-) mice;Atherosclerosis;aorta;RT-PCR;atherosclerosis-related genes

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引用本文：

戴学栋, 尹苗, 荆文, 杜会芹, 叶红燕, 商允菊, 张晾, 邹艳艳, 曲志萍, 潘杰. apoE/LDLR双基因缺失幼龄小鼠主动脉中动脉粥样硬化相关基因的表达[J]. 生理学报 2008; 60 (1): 43-50.

Dai Xuedong, Yin Miao, Jing Wen, Du Huiqin, Ye Hongyan, Shang Yunju, Zhang Liang, Zou Yanyan, Qu Zhiping, Pan Jie. Expressions of atherosclerosis--related genes in aorta in young apoE/LDLR double knockout mice. Acta Physiol Sin 2008; 60 (1): 43-50 (in Chinese with English abstract).