血管生成素样蛋白2促进ApoE^(-/-)小鼠动脉粥样硬化内膜钙化

Angiopoietin-Like 2 Promotes Atherosclerotic Calcification in Aortic Artery ApoE^(-/-) Mice

目的探讨血管生成素样蛋白2(Angptl2)对ApoE-/-小鼠动脉粥样硬化内膜钙化的影响。方法 12只6周龄雄性ApoE-/-小鼠随机分为对照组与干预组,每组6只,对照组小鼠予以高脂饮食,干预组小鼠在高脂饮食的基础上在第8周予以静脉注射人重组Angptl2蛋白,每周一次,持续1个月。两组小鼠高脂饮食喂养至16周龄时处死,测定血清脂质水平,HE染色观察主动脉组织形态学变化;von Kossa染色观察主动脉钙化,测定血管钙含量和碱性磷酸酶活性判断血管钙化程度;分别用免疫组织化学法、Western Blot、实时定量PCR(qRT-PCR)检测血管Runx2(核心结合因子α1)蛋白和mRNA的表达。结果干预组小鼠血清甘油三酯(TG)、总胆固醇(TC)及低密度脂蛋白胆固醇(LDLC)水平显著高于对照组(P<0.05);血管壁Runx2表达水平较对照组显著升高(P<0.05);对照组小鼠主动脉可见粥样硬化斑块,von Kossa染色斑块内未见明显黑色钙盐沉积,而干预组小鼠主动脉HE染色可见内膜较对照组显著增厚,有典型的动脉粥样硬化斑块形成,且von Kossa染色斑块灶状黑色钙化团块较对照组显著增强;干预组小鼠主动脉血管壁钙含量和碱性磷酸酶活性均明显高于对照组(P<0.05)。结论 Angptl2会使高脂饮食ApoE-/-小鼠主动脉血清TG、TC、LDLC水平及Runx2的表达水平增高,同时增加小鼠主动脉中钙含量及血清中碱性磷酸酶活性,促进小鼠主动脉粥样硬化内膜的钙化。Angptl2可促进动脉粥样硬化内膜的钙化,控制和降低血浆中Angptl2的水平似乎可以抑制动脉粥样硬化的钙化,从而为临床预防冠心病的发生发展及治疗提供了一种新的靶标。

Aim To assess the effects of angiopoietin-like 2（ Angptl2） on atherosclerotic calcification in aortic artery of ApoE^- /-mice. Methods Twelve 6-week-old male mice were randomly divided into control group（ n = 6） and interventional group（ n = 6）,the control group were fed with high fat diet and the interventional group were fed with high fat diet and at the eighth week interventional group mice were infused（ intravenously） with purified recombinant Angptl2 once a week for one month. All mice were sacrificed when the mice were 16 weeks old,blood was collected and plasma triglyceride（ TG）,total cholesterol（ TC）,low density lipoprotein cholesterol（ LDLC） were measured,aortic sections were stained with hematoxylin and eosin（ HE） or von kossa and were observed under microscope. Calcium content and alkaline phosphatase activity of aorta were measured to measure the degree of vascular calcification. The expressions of Runx2 protein and mRNA levels in aotic sections of mice were detected by immunohistochemisty,Western Blot and qRT-PCR respectively. Results The plasma TG,TC and LDLC level in interventional group was significantly higher than that in control group and the expression of Runx2 in aortic had the similar results. HE staining demonstrated significant thickening of the intima,with typical atherosclerotic plaque formation in interventional group mice,and von Kossa staining showed spotty black clumps of aortic calcification under the fibrous cap plaque,while control group had atherosclerotic plaques without significant calcium deposits formation; The quantitative analysis showed that aortic vascular wall calcium and alkaline phosphatase activity were significantly higher in the intervention group than that of the control group（ P〈0. 01）. Conclusions Angptl2 could increase ApoE^- /-mice plasma lipid level,it also facilitate the expression of Runx2,calcium content and ALP activity in aortic and then accelerate atherosclerotic calcification. Our experiments demonstrated that Angptl2 could accelerate atherosclerotic calcification. It reminded us that by controlling or decreasing the Anglt2 level in plasma could help inhibit atherosclerotic calcification and then provides a new target to prevent coronary heart disease.