【作者】 张力；

【导师】 朱建华；

【作者基本信息】 浙江大学 ， 内科学， 2002， 硕士

【摘要】 研究背景 血浆同型半胱氨酸(homocysteine,Hcy)水平升高已被证明是动脉粥样硬化(atherosclerosis,AS)独立的危险因素，但Hcy确切的致病机制尚未完全阐明。目前认为AS是一种慢性炎症性疾病，细胞因子在涉及AS各阶段的炎症反应事件中发挥重要的调节作用。白细胞介素-6(interleukin-6,IL-6)是参与AS发病最为密切的细胞因子之一，还可作为血管壁炎症激活的标志物。核转录因子κB(nuclear factor-kappa B,NF-κB)是一种具有双向调节作用的转录因子，最初发现于B淋巴细胞中，近来研究证实在单核细胞、血管内皮细胞和血管平滑肌细胞(vascular smooth muscle cells,VSMCs)中均存在着NF-κB激活通路，它能调控包括IL-6在内的众多炎症介质的基因表达和细胞增殖。NF-κB的异常激活可能是AS发生和发展的始动机制之一，且是机体炎症反应放大与持续的分子生物学机制。新近研究发现Hcy能引起单核细胞IL-6的释放增加，并能诱导细胞内NF-κB的激活，提示Hcy还是一种炎症刺激物。我们推测Hcy的致炎症作用可能为其参与AS发病的重要机制之一。在人类和动物AS病变部位的单核巨噬细胞、内皮细胞和VSMCs中均可检测到IL-6mRNA和蛋白表达，其中VSMCs被认为是血管壁细胞因子最主要的来源。迄今为止，Hcy对VSMCs IL-6表达的作用尚未见文献报道。本研究旨在观察Hcy对大鼠VSMCs IL-6合成和基因表达的影响并深入探讨NF-κB活化在其中的可能调控机制，为进一步明确Hcy致AS的作用机制提供重要的理论依据，同时也为AS发病机制的研究及对其有效防治提供新的思路。 第一部分 同型半胱氨酸对大鼠VSMCs IL-6表达的影响 目的：从核酸和蛋白两个水平观察Hcy对大鼠VSMCs IL-6表达的影响，从而明确Hcy 浙江大学硕土学位论文 是否可以通过刺激VSMCS产生促炎性细胞因子几－6而参与AS的发生。 方法：用组织贴块法原代培养SD大鼠胸主动脉平滑肌细胞；在VSMCS培养基中分别 加入不同浓度的HCy，并孵育不同时间；用酶联免疫吸附（ELISA）法检测细胞培养上清液 中IL－6蛋白含量，用半定量逆转录聚合酶链反应（RTPCR）法检测细胞ILedmRNA的表达。 结果：l、Hey浓度在0刀10．25nunow范围内，其促IL6表达作用呈一定的量效关 系。0刁nunoffe Hey即可增加 IL－6产生，细胞培养上清液中 IL－6含量约为对照的 2．4倍 （p＜0刀5）；0二smmol／L Hey对儿－6产生的刺檄作用更明显，培养液 IL七浓度增至对照的 3．4倍（p＜0．of）。2、0．25mrnoThHcy呈时间依赖关系增加 VSMCs产生 IL－6。细胞培养上 清液中几－6含量在HCy刺激后6h开始高于对照组（P＜0．05），于24h达到高峰（P＜0刀1）， 48h有较明显的下降，但仍高于未受HCy刺激的VSMCS（p＜0刀5），而VSMCS基础表达的 IL－6量不随时间延长而增高。3、RTPCR结果显示：正常状态下VSMCs有低水平的 IL6InRNA表达，Hey可呈剂量依赖关系刺激其表达。0刀1、0刀5、0．l、0．25nunoffe Hey 处理组细胞L－6rnRNA表达量分别为对照的2．39（P＜0．05）、2．38（P＜0．05）、3．33（P＜0．of） 和 3厂1倍（P＜口刀l）。0二smmoUL Hey刺激 VSMCs不同时间后促 IL七rnRNA表达作用呈一 定的时效关系，12h其作用达到高峰，为对照的3．82倍（P＜001）。 第二部分 NF－K B活化在同型半恍氨酸诱导大鼠VSMCS表达几－6中的作用 目的：观察比y对大鼠 VSMCS NF－K B活性的影响及吓－K B活化在仇y诱导的 VSMCS 表达IL－6中的作用，以初步探讨HCy促大鼠VSMCS IL－6表达的细胞内机制。 方法：NF－。B活性检测采用电泳迁移率改变法（electroPhoretic mob小ty shift assay EMSA）；二L6mRNA表达采用RTPCR法检测。 结果：！、应用EMSA技术，可见静息状态下VSMCS具有一定的NF－。B活性水平。经 0 25mmol／L Hey刺激后，NF－。B活性迅速增加。密度扫描仪结果提示，NF－。B活性 30min 时为对照 1．76倍（P＜0．of），lh时为对照 l．gi倍（P（．01），Zh时为对照 l．84倍（P（．01）。 2、Hey刺激 VSMCs IL－6mRNA表达的效应能被 NF－。B特异性抑制剂毗咯咐烷二甲基硫腺 （PDTC）所阻滞（P＜0．05）。 结论 1、H叮呈剂量依赖关系（0．0卜0．25——1几）和时间依赖关系（0－48h），增加大鼠化肌s 2 浙江大学硕土学位论文 几－6蛋白合成和。RNA表达。 2、Hey使大鼠VSMCs NF－。B活性增加。 3、Hey诱导大鼠VSMCs表?更多还原

【Abstract】 Elevated plasma levels of homocysteine (Hey) is an established risk factor for atherosclerotic disease. However, the underlying molecular mechanisms are not fully elucidated. Recent studies suggested that atherosclerosis is an inflammatory process and that cytokines played important roles in this process. Interleukin-6 (IL-6) is a multifunctional proinflammatory cytokine and it could be used as marker of the proinflammatory potential of vascular smooth muscle cells (VSMCs). Nuclear factor- kappa B (NF- K B) is a family of transcription factors that was originally identified in B cells but the novel data showed it was also detectable in VSMCs. NF- K B has been indicated to play a role in the gene expression of inflammatory cytokines and VSMCs proliferation. Abnormal activation of NF- K B is a hallmark of many chronic inflammatory and vascular disease and it also may interfere with initiation of atherosclerotic disease. Hey has been demonstrated to increase IL-6 production in monocyte and induce NF- K B activation in VSMCs, which suggested that Hey may be a inflammatory stimuli. The effect of Hey on the expression of IL-6 in atherognesis and the underling mechanisms are still uncertain till now. We hypothesized that Hey may stimulate IL-6 expression in VSMCs through activation of NF- K B and which may be one of the mechanisms that Hey promote atherogenesis. Part I Effect of homocysteine on IL-6 expression in rat vascular smooth muscle cellsObjective: To determine whether Hey contribute to the pathogenesis of atherosclerosis by increasing the expression of IL-6 mRN A and the production of protein by means of investigating the effect of Hey on rat VSMCs.Methods: Rat VSMCs were stimulated with Hey. Cell ELISA was performed to measure the expression of IL-6 protein. Semiquantitative RT-PCR was used to detect the IL-6 mRN Aexpression.Results: 1. Hey concentration-dependently increased the production of IL-6 protein in rat VSMCs. Compared with control, Hey increased IL-6 production to 2.4-fold at O.lmmol/1 (P<0.05) and 3.4-fold at 0.25mmol/l (P<0.01). 2. Treatment with 0.25mmol/L Hey for 6h began to increase IL-6 production (PO.05), the effect reached maximum at 24h (P<0.01). 3. Hey also significantly enhanced IL-6 mRNA expression with a concentration-dependent and time-dependent pattern. After treating with 0.25mmol/L Hey for 12h, the effect was maximum (PO.01). Hey induced IL-6 mRNA expression in VSMCs to 2.39-fold at O.Olmmol/L (P<0.05), 2.38-fold at 0.05mmol/L (P<0.05^ 3.33-fold at O.lmmol/L (P<0.01) and 3.71-fold at 0.25mmol/LPart II Role of NF- K B on the expression of IL-6 in rat vascular smooth muscle cells induced by homocysteineObjective: To explor the intracellar signalling mechanism, we investigate the effect of Hey on NF- K B activity in VSMCs and role of NF- K B on the expression of IL-6 induced by Hey.Methods: Electrophoretic mobility shift assay(EMSA) was used to examine NF- K B activity. Semiquantitative RT-PCR was used to detect the IL-6 mRNA expression.Results: 1. Hey could rapidly induce the activation of NF- K B in rat VSMCs. Baseline NF- K B nuclear binding in VSMCs increased more than 1.76-fold at 30min(P<0.05^ 1.91-fold at lh(P<0.05) and 1.84-fold at 2h(P<0.05) following stimulation with 0.25mmol/L Hey. 2. PDTC, one of the inhibitor of NF- K B, could significantly inhibit the expression of IL-6 mRNA in VSMCs stimulated by Hcy(P<0.05). Conclution1. Hey significantly increased IL-6 mRNA expression and production in rat VSMCs with a concentration-dependent (0.01-0.25mmol/L) and a time-dependent (0-48h) pattern.2. Hey could induce the activation of NF- K B in VSMCs.3. The effect of Hey on the exprssion of IL-6 in VSMCs is exerted through NF- K B activation pathway.更多还原