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

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

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)双基因缺失(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-1β、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(P<0.05, P<0.01),PDGF-α和TNF-α表达在2 月龄时较同龄WT 小鼠显著上调,除ET-1表达在2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(P<0.05, P<0.01),其中 MCP-1 表达在 2 月龄时达到峰值。NF-κB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-α、IL-1β和 ox-LDL含量均显著高于同龄WT小鼠(P<0.05, 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。

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. β-actin was used as an internal control. Then RT-PCR assay was used to analyze the expression patterns of interleukin-1β （IL-1β）, minor necrosis factor-α （TNF-α）, nuclear factor-κB （NF-κB）, 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-α （PDGF-α）. 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-α, IL- 1β 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- 16, 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-α and TNF-α 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-land ICAM-1 expressions at the age of 3 months were down-regulated to that in WT mice. NF-κB 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-1β expressions were slightly up-regulated at the ages of 2 and 3 months. Serum levels of TC, TG, LDL, HDL, TNF-α IL-1β 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.