纤维细胞在高脂饮食致大鼠动脉粥样硬化形成中的作用

发布时间：2018-01-04 13:23

本文关键词：纤维细胞在高脂饮食致大鼠动脉粥样硬化形成中的作用　出处：《昆明理工大学》2016年硕士论文　论文类型：学位论文

【摘要】：研究背景随着发展中国家生活水平的提高以及西方生活方式的普及,动脉粥样硬化(Atherosclerosis, AS)造成的心血管疾病将是世界范围内最主要的死亡原因。因此,研究动脉粥样硬化形成的原因和机制,对动脉粥样硬化的有效防治具有重要的临床价值和意义。研究发现,肌成纤维细胞在形成动脉粥样硬化斑块中数量增多,产生过量胶原和其他细胞外基质成分,沉积形成纤维帽被认为是动脉硬化斑块和纤维帽形成过程中的重要成分。新近研究表明,纤维细胞是肌成纤维细胞的一个重要来源,参与动脉粥样硬化形成。然而,纤维细胞在动脉粥样硬化中作用的研究报道甚少。本研究从纤维细胞途径,探讨其动脉粥样硬化形成的作用,有助于为动脉粥样硬化的防治提供新的策略和思路。方法动脉粥样硬化SD大鼠模型建立：采用两种高脂饮食(A：基础饲料78.8%,猪油12%,胆固醇3.5%,丙基硫氧嘧啶0.2%,白糖5%,胆酸钠0.5%; B：基础饲料60%,猪油10%,蛋黄粉10%,白糖20%)前后分段喂养SD大鼠,应用全自动生化仪定期监测血脂(TC、TG、HDL-C和LDL-C水平)和HE染色判断腹主动脉斑块形成,确定大鼠动脉粥样硬化模型建成。纤维细胞在动脉粥样硬化中的作用的研究方法：①采用免疫组织化学方法评价纤维细胞(白细胞分化抗原45(cluster of differentiation 45, CD45+ CD34+ Col I+)腹主动脉斑块的存在；②酶联免疫吸附法(ELIS A)观测纤维细胞表面趋化因子配体12 (Chemotaxis cytokine ligand-12, CXCL12)和趋化因子受体4(Chemotaxis cytokine receptor-4, CXCR4) 在AS斑块中的表达,发现CXCL12/CXCR4轴在纤维细胞迁移中的作用；③免疫组织化学和逆转录聚合酶链式反应(RT-PCR)检测平滑肌肌动蛋白α(α-smooth muscle actin, α-SMA+)肌成纤维细胞的表达,以揭示纤维细胞的分化作用；④RT-PCR和蛋白质印迹法(Western Blot)方法,测量正常组和模型组腹主动脉中纤维细胞分泌的细胞外基质(I型胶原蛋白, collagen type I, Col I)、促血管生成因子(platelet-derived growth factor A, PDGF-A)基础金属蛋白酶(matrix metalloproteinase, MMP-2)、促生长因子(transforming growth factor-β, TGF-β)以及趋化因子(monocyte chemoattractant protein-1, MCP-1)的基因和蛋白表达水平,探索纤维细胞在动脉粥样硬化形成过程中发挥的作用。结果(1)两种高脂饮食(联合维生素D3)分段喂养成功建立动脉粥样硬化模型；(2)在正常组和模型组大鼠主动脉上CD45+、CD34+、Col I+三个因子的免疫荧光均呈阳性反应,但模型组中荧光信号强度明显高于正常组,表明模型组大鼠主动脉上纤维细胞的数量明显增加；(3)模型组大鼠主动脉上的荧光强度和基因表达水平明显高于正常组,提示α-SMA+肌成纤维细胞存在于大鼠主动脉斑块上；(4)模型组大鼠主动脉上Col I+、PDGF-A、MMP-2、TGF-β、MCP-1的蛋白和mRNA表达明显高于正常组,表明纤维细胞分泌的这些因子可能在动脉粥样硬化形成中发挥了重要作用。结论(1)两种高脂饲料(联合维生素D3)分段喂养是建立动脉粥样硬化模型一种理想的方法。(2)纤维细胞迁移、分化成为肌成纤维细胞、分泌多种细胞因子(Col I、PDGF-A、MMP-2、TGF-β和MCP-1)在动脉粥样硬化形成中发挥重要作用。这些发现有助于为动脉粥样硬化的防治提供新的策略和思路。
[Abstract]:Background: with the life level of the developing countries and the improvement of the popularity of Western lifestyle, atherosclerosis (Atherosclerosis, AS) caused by cardiovascular disease is the leading cause of death in the world. Therefore, studying the causes and mechanism of the formation of atherosclerosis, has important clinical value and significance of effective prevention and treatment of atherosclerosis. The study found that muscle the number of fiber cell formation in atherosclerotic plaque increased, excessive production of collagen and other extracellular matrix deposition, formation of fibrous cap is the artery plaque and fibrous cap formation is an important component in the process. Recent research shows that muscle fiber cells are an important source of fiber cells, participate in the formation of atherosclerosis. However, research reported effects of fiber cells in atherosclerosis is rare. This study from the fiber cell Way, to explore the role of atherosclerosis, contribute to the prevention and treatment of atherosclerosis to provide new methods and ideas. To establish a method for atherosclerosis SD rat model: using two kinds of high fat diet (A: 78.8% basic feed, 12% lard, 3.5% cholesterol, 0.2% propylthiouracil, 5% sugar, 0.5% sodium cholate; B Foundation: feed 60%, 10% lard, 10% yolk powder, sugar, 20%) before and after feeding SD rats, using automatic biochemical analyzer, regular monitoring of lipid (TC, TG, HDL-C and LDL-C level) and HE staining of abdominal aortic plaque formation, set up a rat model of atherosclerosis. Methods the role of fiber cells in atherosclerosis. The evaluation of the fiber cell immunohistochemical method (leukocyte differentiation antigen 45 (cluster of differentiation 45, CD45+ CD34+ Col I+) abdominal aortic plaque; II ELISA Adsorption method (ELIS A) and observation of fiber cell surface chemokine ligand 12 (Chemotaxis cytokine, ligand-12, CXCL12) and chemokine receptor 4 (Chemotaxis cytokine, receptor-4, CXCR4) expression in AS plaque, found in fiber cell migration in CXCL12/CXCR4 axis; chemical and reverse transcriptase polymerase chain reaction and immunohistochemistry (detection of alpha smooth muscle actin (RT-PCR) -smooth muscle alpha actin, alpha -SMA+) muscle fibroblast cells, to reveal the differentiation of fibroblasts; the RT-PCR and Western blotting (Western Blot method), extracellular matrix fiber cell secretion was measured in normal group and model group in the abdominal aorta (type I collagen, collagen type I, Col I), angiogenic factors (platelet-derived growth factor A PDGF-A (matrix) based metalloproteinase metalloproteinase, MMP-2 (trans), growth factor Forming growth factor- TGF- beta, beta chemokine (monocyte) and chemoattractant protein-1, MCP-1) the expression level of gene and protein, explore the fiber cell formation process play a role in atherosclerosis. Results (1) two kinds of high fat diet (combined vitamin D3) segment feeding atherosclerosis model was successfully established; (2) CD45+ in the normal group and model group, rat aortic CD34+, immunofluorescence three factor Col I+ was positive in the model group, but the fluorescence signal intensity was significantly higher than the normal group, model group rats showed that aortic number of fibroblasts increased significantly; (3) the fluorescence intensity and the gene of aorta of rats in the model group the expression level was significantly higher than the normal group, suggesting that alpha -SMA+ fibroblasts in rat aortic plaque; (4) the aorta of rats in the model group Col I+, PDGF-A, MMP-2, TGF- beta, MCP-1. The white and the expression of mRNA was significantly higher than the normal group, showed that the fiber cells secrete these cytokines may play an important role in the formation of atherosclerosis. Conclusion (1) two kinds of high fat diet (vitamin D3) segmented feeding is the model of atherosclerosis was established an ideal method. (2) cell migration, differentiation into muscle fibroblasts secrete a variety of cytokines (Col, I, PDGF-A, MMP-2, TGF- beta and MCP-1) play an important role in the formation of atherosclerosis. These findings provide new methods and ideas in the prevention of atherosclerosis.

【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R543.5

【相似文献】