【作者】 李晶；

【导师】 夏豪；

【作者基本信息】 武汉大学 ， 内科学， 2017， 硕士

【摘要】 1.目的:用高脂饮食诱导的OSMR-β、ApoE全身基因双敲除(OSMR-β-/-ApoE-t-)、ApoE全身基因敲除(ApoE-/-)小鼠和小鼠体内分离、体外氧化修饰低密度脂蛋白刺激的腹腔巨噬细胞作为实验对象,探讨OSMR-β缺乏在高脂饮食诱导的小鼠动脉粥样硬化模型和氧化修饰低密度脂蛋白刺激下体外巨噬细胞泡形成沫细胞的可能机制。2.方法:(1)动物分组:将实验动物分为4组,分别为ApoE-/-正常饮食组(ApoE-/-NC)、ApoE-/-高脂饮食组(ApoE-/-HFD)、OSMR-β-/-ApoE-/-正常饮食组(OSMR-β-/-AβoE-/-NC)、OSMR-β-/-ApoE-/-高脂饮食组(OSMR-β-/-AβoE-/-HFD)。(2)建立动脉粥样硬化模型及取材:高脂饮食喂养28周后用巴比妥钠麻醉,打开胸腔游离出整个主动脉树(从主动脉弓起始处到髂动脉左右分支处),剥除血管外膜脂肪、拍照、固定。剪下行主动脉树拍照后的心脏,切下心尖下1/3,保留上1/3主动脉窦。(3)小鼠心脏、血管的病理取材样本用于石蜡包埋切片,采用油红O染色评价全长主动脉的粥样斑块表面积,H&E染色评价动脉粥样硬化斑块横截面积及坏死中心核的面积,PSR染色评价斑块横截面积大小及斑块内胶原含量,荧光染色用于确定斑块内OSMR-β的表达及细胞定位或其它种类细胞或因子的含量。(4)小鼠动物实验中的分生样本用于检测OSMR-β表达水平、炎症因子转录及表达水平、信号通路相关因子的水平。(5)腹腔原代巨噬细胞培养:从OSMR-β-/-AβoE-/-和AβoE-/-小鼠腹腔洗脱出巨噬细胞,以血清饥饿法处理24小时后在体外给予ox-LDL孵育24小时,固定,随后用油红O染色探测巨噬细胞脂质积聚情况。(6)骨髓移植:雄性ApoE-/-小鼠首先经总剂量为11 Gy的放射性照射,随后将分别从OSMR-β-/-ApoE-/-和ApoE-/-小鼠体内提取的骨髓注射入受体小鼠体内,经16周的HFD后进行相关的病理染色及分子生物学和体外刺激实验。(7)人体标本:从经心脏移植术中取出的冠心病患者心脏分离出右冠状动脉进行相关的分子生物学实验,检测OSMR-β在血管内的表达量及细胞定位。3.结果:(1)OSMR-β与Mac3荧光双染显示OSMR-β在动脉粥样硬化斑块中主要定位于巨噬细胞中,Western blot分析及荧光双染半定量均显示在冠心病人以及小鼠的斑块内及体外培养的原代巨噬细胞中其含量均升高。(2)油红O染色以及H&E染色表明敲除OSMR-β的OSMR-β-/-(ApoE-/-背景)减轻高脂饮食诱导的动脉粥样硬化形成,主动脉窦及头臂干动脉的斑块面积均缩小。(3)敲除OSMR-β帮助维持动脉粥样硬化斑块的稳定性,体现在OSMR-β-/-ApoE-/-组斑块内坏死核面积缩小(主动脉窦及头臂干动脉H&E、PSR染色)、胶原含量增加(PSR染色)、平滑肌细胞含量增多(SMA免疫荧光染色)、脂质沉积(油红O染色)和巨噬细胞浸润减少(CD68荧光染色)。(4)敲除OSMR-β起到的抗动脉粥样硬化作用主要依赖其抗炎效应,实时PCR显示促炎症因子的转录水平降低,ELISA检测到血浆中促炎症因子的含量降低。(5)敲除OSMR-β通过阻断JAK/STAT3信号通路抑制及蛋白质表达水平,Western blot法显示JAK2、STAT3的磷酸化水平下调,荧光双染结果显示巨噬细胞内p-STAT3含量降低。(6)腹腔原代巨噬细胞体外培养试验和骨髓移植试验与上述实验结果一致并进一步巩固了上述结论,由骨髓移植诱导的获得性巨噬细胞OSMR-β缺乏可改善动脉粥样硬化,抑制泡沫细胞形成——可能与促进巨噬细胞的LDL转运平衡偏向于向胞外运输有关。更多还原

【Abstract】 Objective:High-fat-diet(HFD)induced systemic OSMR-β-/-ApoE-/-(Double KO),ApoE-/-(ApoE knockout)mice and isolated primary peritoneal macrophages under ox-LDL stimulation were used as experimental subjects to explore the underlying mechanism of OSMR-β deficiency on the development of HFD induced atherosclerosis of mouse models both in vivo and in vitro.Methods:(1)Experimental mouse were randomly separated into four groups,which were ApoE-/-NC,ApoE-/-HFD,OSMR-β-/-ApoE-/-NC and OSMR-β-/-ApoE-/-HFD respectively.(2)Male mouse of 8-week-old were fed with HFD or NC for up to 28 weeks before sacrificed.After anesthetization with pentobarbital Sodium,open the chest and rapidly separate the entire aorta(from the root of aorta to the branch of iliac artery),followed by dissection of adipose tissue from the vascular adventitial,take photos and then fixed in formalin.The lower third cardiac apex were removed and the upper third were reserved to obtain the aortic sinus.(3)Paraffin-embedded tissue slices were used to perform pathological and immunofluorescent staining.Oil Red O staining was performed to analyze the en face atherosclerotic lesions of the aorta.The aortic roots and brachiocephalic arteries were stained with hematoxylin and eosin(H&E)for morphological analysis of atherosclerotic plaques,picrosirius red(PSR)to evaluate collagen deposition.Immunofluorescence were performed to detect the expression of OSMR-β within the plaque,transcriptional and protein levels of pro-inflammatory cytokines,activated levels of relative signaling transducers.(4)Studies of primary peritoneal macrophages:Bone marrow-derived macrophages eluted from the peritoneum of ApoE-/-mice were harvested under sterile conditions.After serum starvation for 24 h,the macrophages were incubated with 15μg/ml Ox-LDL for 24 h.Finally,the cells were fixed and stained with Oil O Red to asses the lipid accumulation capacity of macrophages.(5)Bone marrow transplantation:Male ApoE-/-recipient mice were irradiated with a total dose of 11 Gy prior to transplantation.Donor bone marrow was isolated from male OSMR-β-/-ApoE-/-or ApoE-/-mice by flushing femurs and tibias and injected to each irradiated mouse.After a HFD for 16 weeks,the mice were sacrificed for the following experiments.(6)Human specimens:Atherosclerotic plaques were collected from the right coronary artery of patients with CHD who undergoing heart transplantation.Control samples were obtained from normal donors who were unsuitable for transplantation without cardiovascular diseases.Vascular tissues were under procedures similar to animal operation to detect the expression and location of OSMR-β in the artery.Results:(1)Double-immunofluorescence staining with OSMR-β and Mac3 revealed revealed an increased OSMR-P expression,which located mainly in the macrophages of the atherosclerotic lesions of patients with CHD and the HFD-induced ApoE-/-mouse model.(2)En face analysis of atherosclerotic lesions by Oil Red O and H&E staining indicat ed that OSMR-β deficiency(ApoE-/-background)attenuated the development of HFD induced atherosclerosis,plaque areas of aorta root and brachiocephalic arteries were both decreased.(3)OSMR-β deficiency enhanced the stability of atherosclerotic plaques,presented as total necrotic core area was significantly smaller in OSMR-β-/-ApoE-/-mice(H&E staining of aorta root and brachiocephalic arteries),the collagen and SMC content covering the fibrous cap were increased(PSR and immunofluorescent staining),lipid accumulation decreased(Oil O Red staining)and macrophage infiltration reduced(immunofluorescent staing with anti-CD68).(4)The protective role of OSMR-β deficiency in atherogenesis was dependent on its anti-inflammation effect,Real-time PCR analysis showed that multiple pro-inflammatory genes were significantly decreased.The level of inflammatory factors in the serum was also down regulated,measured with ELISA kit.(5)Loss of OSMR-β inhibited the JAK2/STAT3 signal pathway.Western blot showed the phosphorylated level of both factors were decreased with the immunofluorescent staining revealed the activation of STAT3 were clearly impaired.(6)In vitro experiments of primary peritoneal macrophages stimulation with ox-LDL were consistent with in vivo results.Bone marrow transplantation further confirmed the conclusion by displaying an acquired loss of OSMR-β improved the HFD induced atherosclerosis with reduction of foam cell formation which was possibly attributed to the intervention of LDL flux of macrophages by facilitating its extracellular transportation.更多还原