NARC-1蛋白在新西兰兔动脉粥样硬化病变中的表达
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湖南省科技计划重点项目(2008FJ2006);动脉硬化学湖南省高校科技创新团队经费资助


Investigation of NARC-1 Protein Expression in Atherosclerotic Lesion of New Zealand Rabbit
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    摘要:

    目的探讨NARC-1在新西兰兔动脉粥样硬化病变血管壁中的表达。方法健康纯种雄性新西兰白兔16只,适应性喂养1周后,随机分为对照组和高胆固醇组,每组8只;对照组给予普通颗粒饲料喂养,高胆固醇组给予高胆固醇饲料(2%胆固醇)喂养;两组动物于8周末安乐处死。全自动生化分析仪测定兔血浆中甘油三酯、总胆固醇、高密度脂蛋白胆固醇和低密度脂蛋白胆固醇含量,计算动脉粥样硬化指数、高密度脂蛋白胆固醇/低密度脂蛋白胆固醇比值。苏丹Ⅳ染色测量兔主动脉粥样硬化病变面积。油红O染色及HE染色观察兔主动脉病理组织学改变,并进行病理形态学定量分析。免疫组织化学法与免疫印迹法检测兔主动脉组织NARC-1蛋白的分布及表达水平。结果8周末,高胆固醇组兔血浆总胆固醇、高密度脂蛋白胆固醇、低密度脂蛋白胆固醇和、动脉粥样硬化指数水平明显升高,而高密度脂蛋白胆固醇/低密度脂蛋白胆固醇比值明显降低。高胆固醇组出现了明显的动脉粥样硬化病变,主动脉内膜面粥样硬化病变面积为27.41%±2.12%,与对照组(0.51%±0.20%)相比差异显著(P<0.05)。病理组织学显示高胆固醇组兔主动脉内膜明显增厚,内膜/中膜厚度比明显增大;油红O染色光镜下显示,高胆固醇组兔主动脉内皮下斑块内泡沫细胞呈橘红色,对照组主动脉内膜未见明显着色;HE染色光镜下显示,高胆固醇组主动脉内膜显著增生,形成明显斑块,斑块中纤维组织增生,可见大量泡沫细胞,胞核较小,胞浆内含有大量脂质空泡,对照组内膜薄且结构完整。免疫组织化学检测发现高胆固醇组兔主动脉NARC-1蛋白表达明显,染色呈强阳性,且群集于内膜斑块处;在高倍镜下,NARC-1蛋白主要分布在泡沫细胞的胞浆和胞膜,而在对照组兔血管壁组织中未见表达。免疫印迹检测发现高胆固醇组兔主动脉NARC-1蛋白呈高表达,而对照组兔血管壁组织未见表达。结论新西兰兔的主动脉粥样硬化病变处NARC-1蛋白表达明显,其主要分布在泡沫细胞的胞浆和胞膜中,正常对照组兔主动脉血管壁组织未检测到NARC-1蛋白。提示NARC-1/PCSK9是影响动脉粥样硬化病变形成与发展的重要因素。

    Abstract:

    Aim To examine the expression of NARC-1 protein in atherosclerotic lesion of New Zealand rabbits. Methods Male purebred New Zealand rabbits with adaptation feeding for 1 week,were randomly divided into control group(n = 8) and high-cholesterol group(n = 8).The control group was given a normal control diet and the high-fat group was fed a high-cholesterol diet(2%cholesterol).All rabbits were euthanized at the end of the 8-week experiment. Plasma triglyceride(TG),total cholesterol(TC),high density lipoprotein cholesterol(HDLC) and low density lipoprotein cholesterol(LDLC) were determined by commercially enzymatic methods.Atherosclerosis index(AI) and the HDLC/LDLC ratio were calculated.SudanⅣstaining was used to examine the aortic atherosclerotic lesions.The pathological changes of rabbit aortic preparations was quantified by computer-assisted image analysis of oil red O and HE staining using Image-Pro Plus image analysis software.NARC-1 protein distribution and expression was analyzed by immunohistochemistry and Western blotting.Results Plasma TC,HDLC and LDLC concentrations,and AI were markedly increased, but the HDLC/LDLC ratio was markedly decreased in high-cholesterol group.High-cholesterol diet induced significant aortic atherosclerotic lesions.Histopathology analysis showed that aortic intima was obviously thickened and the intima/media thickness ratio was significantly increased in high-cholesterol group rabbits.Oil red O staining indicated that the number of foam cells in artery endothelium were markedly increased in the high-cholesterol group compared with the control group.HE staining revealed the aortic intima in control rabbits was intact and thin,compared to thickened aortic intima and remarkable atherosclerotic plaques in high-fat group rabbits.Immunohistochemical detection showed NARC-1 protein was found in the intima plaque and located in cytoplasma and plasma membrane of foam cells in high-fat group. Western blotting analysis also indicated NARC-1 protein expression was significantly elevated in the aortic atherosclerotic plaque of high-fat group rabbits.Conclusion The high-cholesterol diet induced aortic atherosclerotic lesion in New Zealand rabbits.NARC-1 protein was remarkably expressed in aortic atherosclerotic plaque and located in the cytoplasma and plasma membrane of the foam cells in high-fat group rabbits.The results indicate that NABC-1/PCSK9 is one of the important factors involved in atherosclerosis development.

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唐志晗,刘录山,彭娟,杨琼,刘慧婷,姜志胜. NARC-1蛋白在新西兰兔动脉粥样硬化病变中的表达[J].中国动脉硬化杂志,2009,17(6):439~443.

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