基于斑块稳定性对比青年与老年ApoE^(-/-)小鼠冠状动脉粥样硬化的特点

Based on the plaque stability to explore the characteristics of atherosclerosis between young and old ApoE^(-/-)mice

目的建立青年及老年载脂蛋白E基因敲除(ApoE^(-/-))小鼠动脉粥样硬化模型,探讨青年与老年小鼠动脉粥样硬化斑块稳定性的差异及存在差异的可能机制。方法采用8周龄和32周龄ApoE^(-/-)雄性小鼠分别作为青年模型(QM)组和老年模型(LM)组,相同遗传背景和匹配周龄的C57BL/6J雄性小鼠分别作为青年对照(QC)组和老年对照(LC)组。模型组给予高脂饲料,对照组则给予普通饲料。干预28 w后,行小动物超声、油红O染色和苏木素-伊红(HE)染色,观察冠状动脉斑块的形态学;微板法检测小鼠血脂水平;酶联免疫吸附试验(ELISA)检测血清趋化因子单核细胞趋化蛋白(MCP)-1水平;普鲁士蓝染色观察冠状动脉斑块内出血情况;Masson染色计算冠状动脉斑块内胶原纤维含量;免疫组化染色观察冠状动脉斑块内CD68、基质金属蛋白酶(MMP)9表达;RT-q聚合酶链反应(PCR)和免疫印迹法检测主动脉细胞间黏附分子(ICAM)-1及血管细胞黏附分子(VCAM)-1 mRNA和蛋白表达情况。结果小动物超声观察可见模型组主动脉弓部及颈动脉部均有明显斑块。与对照组相比,同年龄段模型组血清三酰甘油(TG)、总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)水平显著升高(P<0.05)。与QM组相比,LM组胶原纤维含量减少,斑块CD68及MMP9含量升高,普鲁士蓝染色阳性颗粒数量减少,差异有统计学意义(P<0.05,P<0.01)。与对照组相比,同年龄段模型组ICAM-1、VCAM-1 mRNA和蛋白表达均明显上调(P<0.01,P<0.05)。LM与QM组相比,ICAM-1 mRNA和蛋白、VCAM-1 mRNA表达明显上调(P<0.05)。结论在相同周期的高脂饮食条件下,青年与老年ApoE^(-/-)小鼠动脉粥样硬化病程不同,老年ApoE^(-/-)小鼠冠状动脉粥样硬化斑块的不稳定性主要与炎症反应、细胞外基质降解有关,而青年ApoE^(-/-)小鼠冠状动脉粥样硬化斑块的不稳定性则主要与斑块内出血密切相关。

Objective To establish young and old apolipoprotein E gene knockout(ApoE^(-/-))mice models of atherosclerosis to investigate the variations in the stability of atherosclerotic plaques between young and old mice and the underlying mechanisms behind these differences.Methods 8-week-old and 32-week-old male mice with ApoE^(-/-)were used as the young model(QM)group and old model(LM)group,respectively.The young control(QC)group and old control(LC)group consisted of C57BL/6J male mice with the same genetic background and matched weeks.The model groups were given high-fat feed,while the control groups were given normal feed.After 28 w of intervention,morphology of coronary plaques was observed by small animal ultrasound,Oil red O staining,and hematoxylin-eosin(HE)staining.Blood lipid level in mice was detected by microplate method.Enzyme-linked immunosorbent assay(ELISA)was used to detect the level of serum chemokine monocyte chemoattractant protein(MCP)-1.Prussian blue staining was used to observe intracoronary plaque hemorrhage.Collagen fiber content in coronary artery plaques was calculated after Masson staining.Immunohistochemical staining was used to observe the expression of CD68 and matrix metalloproteinase(MMP)9 in coronary artery plaques.RT-q polymerase chain reaction(PCR)and Western blot were used to detect the expressions of intercellular adhesion molecule(ICAM)-1 and vascular cell adhesion molecule(VCAM)-1 mRNA and protein in aortic tissue.Results Ultrasonic observation of small animals showed that there were obvious plaques in the aortic arch and carotid artery in the model group.Compared with the control group,the levels of serum triacylglycerol(TG),total cholesterol(TC),and low-density lipoprotein cholesterol(LDL-C)in same age model group were significantly increased(P<0.05).Compared with the QM group,the collagen fiber contents in the LM group was decreased,the plaque CD68 and MMP9 contents were increased,and the number of Prussian blue-stained positive particles was decreased,and the difference was statistically significant(P<0.05,P<0.01).Compared with the control group,the expressions of ICAM-1,VCAM-1 mRNA and protein in same age model group were significantly up-regulated(P<0.01,P<0.05).Compared with QM group,the expressions of ICAM-1 mRNA and protein and VCAM-1 mRNA were significantly up-regulated in LM group(P<0.05).Conclusions Under the same cycle of high-fat diet condition,the courses of atherosclerosis in young and old ApoE^(-/-)mice are different,and the instability of coronary atherosclerotic plaques in old ApoE^(-/-)mice is mainly related to inflammatory response and extracellular matrix degradation,while the instability of coronary atherosclerotic plaques in young ApoE^(-/-)mice is mainly closely related to intraplaque hemorrhage.