SARAF蛋白对动脉粥样硬化的作用及机制研究

发布时间：2018-04-22 04:10

  本文选题：动脉粥样硬化 + 血管紧张素Ⅱ　； 参考：《西南医科大学》2017年硕士论文

【摘要】：目的:建立ApoE基因敲除大鼠(Apolipoprotein E-deficient Rats,ApoE~(-/-)Rats)动脉粥样硬化模型及培养大鼠巨噬细胞,探讨钙库操作性Ca~(2+)内流调节因子(Store-Operated Calcium Entry Associated Regulatory Factor,SARAF)在动脉粥样硬化中的作用及机制。方法:第一部分:建立ApoE基因敲除大鼠动脉粥样硬化模型将8周龄ApoE基因敲除大鼠共16只,分为实验组和对照组,予高脂饲料喂养12周。实验组在干预的前4周予2004型Alzet渗透泵植入大鼠皮下,内部填充血管紧张素Ⅱ(AngiotensinⅡ,AngⅡ),浓度为25mg/ml,以0.25μl/hr的速率稳定释放28天。对照组仅予等量生理盐水。检测血压、心率、体重,12周后麻醉取血并处死,检测炎症因子白细胞介素-1β(Interleukin-1β,IL-1β)、肿瘤坏死因子-α、(Tumor necrosis factor-α,TNF-α),血脂TC、TG、HDL、LDL;取心脏及主动脉组织制备病理切片,作HE、油红O、Masson染色,评估动脉粥样硬化斑块情况;采用实时定量基因扩增荧光检测系统(q PCR)以及蛋白免疫印迹法(western-blotting)检测主动脉SARAF、TNF-α、IL-1β表达。第二部分:SARAF蛋白对巨噬细胞炎症因子的调节作用将NR8383巨噬细胞分为对照组(Blank),ox-LDL组,空病毒干扰组(NC+ox-LDL),SARAF过表达慢病毒载体转染组(SARAF+ox-LDL)。分别以空载体慢病毒和过表达SARAF载体慢病毒,转染NC+ox-LDL组和SARAF+ox-LDL组各24 h,ox-LDL(终浓度100μg/ml)干预ox-LDL组、NC+ox-LDL组和SARAF+ox-LDL组各24 h,采用western blotting技术检测各组SARAF、TNF-α、IL-1β表达。再将NR8383巨噬细胞分为对照组(Blank),ox-LDL组,空病毒载体干扰组(NC+ox-LDL),si-SARAF慢病毒干扰载体组(si-SARAF+ox-LDL)。分别以空载体慢病毒和si-SARAF干扰载体慢病毒,转染NC+ox-LDL组和si-SARAF+ox-LDL组各24 h,ox-LDL(终浓度100μg/ml)干预ox-LDL组、NC+ox-LDL组和si-SARAF+ox-LDL组各24 h,western blotting检测SARAF、TNF-α、IL-1β表达。结果:1.成功建立ApoE基因敲除大鼠动脉粥样硬化模型,大鼠主动脉根部粥样斑块明显;2.实验组ApoE基因敲除大鼠的主动脉SARAF蛋白及炎症因子TNF-α、IL-1β表达升高;3.SARAF过表达组NR8383巨噬细胞炎症因子IL-1β、TNF-α表达增加;4.SARAF基因干扰组NR8383巨噬细胞IL-1β、TNF-α表达降低。结论:1.血管紧张素Ⅱ诱导可成功建立ApoE基因敲除大鼠动脉粥样硬化模型,SARAF可能与动脉粥样硬化有关;2.SARAF可能通过增加巨噬细胞炎症因子IL-1β、TNF-α表达,促进动脉粥样硬化斑块形成。
[Abstract]:Aim: to establish the atherosclerosis model of Apolipoprotein E-deficient ratssis-ratsl in ApoE knockout rats and to investigate the role and mechanism of Store-Operated Calcium Entry Associated Regulatory factorial SARAF in atherosclerosis, and to investigate the role of Store-Operated Calcium Entry Associated Regulatory Regulatory SARAF in atherosclerosis. Methods: in the first part, 16 8-week-old ApoE knockout rats were divided into experimental group and control group, and fed with high-fat diet for 12 weeks. The experimental group was subcutaneously implanted with 2004 Alzet osmotic pump in the first four weeks of intervention, and was filled with angiotensin 鈪，

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