文章摘要
张友明,杨 笛,吴恒芳,卞智萍,徐晋妉,顾春荣,陈相健,王连生.低剪切力下调PPAR信号通路参与BALB/c小鼠局部血管早期病变的实验研究[J].南京医科大学学报,2015,(5):607~614
低剪切力下调PPAR信号通路参与BALB/c小鼠局部血管早期病变的实验研究
Au experimental study of low shear stress down-regulated PPAR signaling pathway involved in early local vascular lesions of BALB/c mice
投稿时间:2015-01-17  
DOI:10.7655/NYDXBNS20150503
中文关键词: 低剪切力  动脉粥样硬化  PPAR 信号通路  中膜增厚  内皮细胞损伤
英文关键词: low shear stress  atherosclerosis  PPAR signaling pathway  media thickening  endothelial cell damages
基金项目:国家自然科学基金资助(81170220)
作者单位
张友明 南京医科大学第一附属医院分子心脏病室,心内科,江苏 南京 210029 
杨 笛 南京医科大学第一附属医院心内科,江苏 南京 210029 
吴恒芳 南京医科大学第一附属医院心内科,江苏 南京 210029 
卞智萍 南京医科大学第一附属医院分子心脏病室,江苏 南京 210029 
徐晋妉 南京医科大学第一附属医院心内科,江苏 南京 210029 
顾春荣 南京医科大学第一附属医院分子心脏病室,江苏 南京 210029 
陈相健 南京医科大学第一附属医院分子心脏病室,江苏 南京 210029 
王连生 南京医科大学第一附属医院心内科,江苏 南京 210029 
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中文摘要:
      目的:研究局部血流低剪切力对BALB/c小鼠局部血管内皮细胞损伤及平滑肌细胞增殖的影响及其分子机制。方法:以左侧颈总动脉套管植入术造成局部血流剪切力变化。30只雄性BALB/c小鼠随机分为假手术组(n = 10,普通饮食)?模型1组(n = 10,套管植入术+普通饮食)?模型2组(n = 10,套管植入术+高脂饮食)。术后喂养8周,监测小鼠体重,颈总动脉超声测量术后3 d和术后8周左?右颈总动脉血流峰值?血管舒张末内径及剪切力变化,检测血脂水平,HE染色观察颈总动脉病理形态学变化,测量血管中膜厚度,透射电镜观测颈总动脉内皮细胞?内皮下超微结构。芯片比较小鼠低剪切力区与自身正常剪切力区血管段基因表达谱变化,GO及KEGG信号通路分析与细胞迁移及增殖相关基因和信号通路的变化并行实时定量PCR(quantificational real time-polymerase chain reaction,QRT-PCR)验证。结果:与假手术组相比,模型1组小鼠术后8周体重及血脂均无明显差异(P > 0.05),模型2组小鼠术后8周体重增加明显,总胆固醇(TC)?甘油三酯(TG)?低密度脂蛋白胆固醇(LDL-C)及高密度脂蛋白胆固醇(HDL-C)水平均升高(P < 0.05)。模型1?2组左侧颈总动脉套管近心端呈低剪切力变化,HE染色可见内皮细胞不同程度损伤,血管中膜增厚,无脂纹和斑块形成;电镜下可见有不同程度的内皮细胞空泡化形成,内皮下未见脂质空泡和脂滴堆积。合并高脂喂养加强了低剪切力导致的血管病变。芯片检测提示与自身正常剪切力血管段相比,低剪切力血管段标本呈现miR-27a上调(>2倍),PPAR信号通路下调,MAPK?TGF-β?PI3K/Akt及NF-κB信号通路上调,包括TGFB2?END1?CTGF等促细胞迁移及(或)增殖的基因上调 > 2.0倍。QRT-PCR验证结果显示:相比于自身正常血管段,在低剪切力血管段中PPARG下调 0.14倍,TGFB2和EDN1分别上调 2.51倍和1.83倍,差异有统计学意义(P < 0.05,n = 4),与基因芯片结果相符。结论:BALB/c小鼠颈总动脉套管植入术后8周,与正常剪切力血管段相比,低剪切力血管段局部见内皮细胞损伤及中膜增厚,提示局部低剪切力变化是血管动脉粥样硬化早期病变的因素,可能与其血管壁局部PPAR信号通路下调后引起的促细胞增殖?迁移相关基因表达及信号通路激活有关。
英文摘要:
      Objective:To study the influence and molecular mechanisms of low shear stress on local vascular endothelial injury and smooth muscle cell proliferation of BALB/c mice. Methods:Thirty BALB/c mice were randomly divided into three groups. Ten mice were taken as the sham-operated group and fed in a normal diet. The model one and model two were established by rapid perivascular collar placement at left common carotid artery(LCCA)and fed in a normal and a high-fat diet,respectively. The body weight was measured periodically. On the third day and eighth week post-operation,the alterations of peak blood flow,end-diastolic diameter of vessels and shear stress of carotid artery were assessed by carotid artery doppler ultrasound. The variations of lipid metabolism were detected by automatic biochemical analyzer during the eighth week post-operation. The pathomorphological and ultrastructural changes of carotid artery were evaluated by hematoxylin-eosin staining and electron microscope,respectively. The gene expression profile changes in the low shear stress blood vessels and in the self-controlled normal shear stress blood vessels were compared by microarray,and then verified by (quantificational real time- polymerase chain reaction,QRT-PCR). GO and KEGG signaling pathway were analyzed and focused on the changes of genes and signaling pathways related to cell migration and proliferation. Results:Compared with sham-operated group,the body weight and serum lipids levels had no significant differences(P > 0.05) in group model one fed with normal diet at the eighth week post-operation. However,the body weight and serum lipids(TC,TG,LDL-C and HDL-C)significantly increased(P < 0.05) in group model two. The shear stress of left common carotid artery-proximal (LCCA-P)was low in group model one and model two. Meanwhile,there were endothelial cell damages,media thickening and endothelial cell vacuolation in the LCCA-P of both group model one and group model two. The lesions in group model two were more serious than those in group model one. But no fatty streaks,plaque formations and lipid accumulations were observed by light microscope and electron microscope. Microarray results suggested that compared with the self-controlled blood vessels from normal shear stress,the blood vessels from low shear stress presented a remarkable miR-27a expression elevation(> 2.0-fold),PPAR signaling pathway down-regulation,MAPK,TGF-β,PI3K/Akt and NF-κB signaling pathway up-regulation,and the cell migration and / or proliferation-related genes up-regulation(TGFB2,END1,CTGF,etc.). The results of QRT-PCR were consistent with microarray. The expression of PPARG decreased to 0.14-fold(P < 0.05,n = 4),TGFB2 and EDN1 increased to 2.51-fold and 1.83-fold,respectively(P < 0.05,n = 4)in the blood vessels from low shear stress. Conclusion:Compared with the blood vessels from normal shear stress,the blood vessels from low shear stress showed the damage of local endothelial cells and media thickening after eight weeks post-operation in BALB/c mice. These changes may be associated with the down-regulation of local PPAR signaling pathways,thereby activating the genes expression and signaling pathways related to cell proliferation and migration.
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