氧化低密度脂蛋白损伤血管内皮细胞及促血管平滑肌细胞增殖的机制

目的 观察氧化低密度脂蛋白(ox-LDL)诱导人脐静脉血管内皮细胞株(ECV304)损伤及损伤条件培养基作用于血管平滑肌细胞(VSMC)的影响.方法 采用流式细胞术(FCM)测定ECV304细胞内Ca2+、线粒体膜电位(MMP)、凋亡率及VSMC细胞周期各时相DNA含量、增殖指数.结果 ox-LDL致ECV304细胞活力降低,细胞内Ca2+浓度升高,MMP下降,细胞凋亡率明显增高；损伤条件培养基有明显促进VSMC增殖,与正常对照组比较有显著差异(P＜0.01,P＜0.001).结论 ox-LDL导致血管内皮细胞损伤,引起ECV304细胞内Ca2超载,致线粒体功能障碍,促进细胞凋亡；促进VSMC增殖.     

氧化低密度脂蛋白诱导血管内皮细胞损伤的条件培养基对血管平滑肌细胞增殖的作用

目的观察氧化低密度脂蛋白（OX—LDL）损伤血管内皮细胞（VEC）的条件培养基对血管平滑肌细胞（VSMC）生长状态的影响。方法在培养的大鼠胸主动脉平滑肌细胞上，分别采用MTT染色法、流式细胞术及增殖细胞核抗原免疫组化方法等，检测OX—LDL诱导VEC损伤的条件培养基对VSMC生长率、细胞周期及PCNA表达的影响。结果OX—LDL可呈浓度依赖性损伤VEC的形态、减少上清液中NO含量、升高上清液中ET-1含量。终浓度为1001μg,／ml的ox-LDL与VEC共同孵育24h制备的条件培养基，能明显促进VSMC生长、提高PCNA表达及增加细胞周期S期细胞数。结论血管内皮细胞受到OX—LDL损伤时，制备的条件培养基能促进血管平滑肌细胞增殖，这可能是氧化低密度脂蛋白致动脉粥样硬化形成的原因之一。     

芦丁抑制氧化低密度脂蛋白诱导的血管平滑肌细胞增殖

目的:探讨芦丁在氧化低密度脂蛋白(ox-LDL)诱导的血管平滑肌细胞(VSMC)增殖中的作用。方法:制备ox-LDL,采用贴壁法分离培养C57BL/6J小鼠的VSMC,将细胞分为对照组、芦丁组、ox-LDL组和芦丁+ox-LDL组,MTT法检测各组VSMC细胞增殖活性,Western blot检测VSMC中磷酸化细胞外信号调节激酶(p-ERK)的表达情况。结果:芦丁组与对照组细胞增殖活性及p-ERK蛋白表达水平均无明显差异(P均0.05);ox-LDL组细胞增殖活性及p-ERK蛋白表达水平均显著高于对照组(P均0.05);与ox-LDL组相比,芦丁+ox-LDL组细胞增殖活性及p-ERK蛋白表达水平均明显降低(P均0.05)。结论:芦丁可能通过抑制ERK信号通路,抑制ox-LDL诱导的VSMC的增殖。     

氨氯地平抑制氧化修饰低密度脂蛋白诱导的血管平滑肌细胞增殖

许多研究表明 ,血管中膜来源的平滑肌细胞 (VSMC)移行到内膜异常增殖和积聚在动脉粥样硬化 (AS)的发生和发展中起了早期和关键的作用。已知有许多因素能促进VSMC的增殖和移行 ,例如 ,血小板源生长因子 (PDGF)、成纤维细胞生长因子、血清、凝血酶血管紧张素Ⅱ等。近年来还发现 ,氧化修饰的脂蛋白 ,特别是低密度脂蛋白 (LDL)经氧化修饰后致AS的作用增强。从VSMC的生长 /增殖的机制来说 ,细胞内游离钙浓度具有很大的调控能力 ,在这种类型的细胞中 ,电压依从式钙离子通道在细胞内Ca2 + 平衡中起了重要的作用。本实验选用氧化修饰低密…     

血脂康及氧化低密度脂蛋白对牛血管平滑肌细胞增殖的影响

他汀类调脂药除调节血脂、影响动脉粥样硬化 (ＡＳ)的发展过程外 ,尚对ＡＳ形成有直接的抑制作用。我们于 1999年 1～ 4月观察了国产含他汀类成分的调脂药血脂康和氧化低密度脂蛋白 (ＯＸ ＬＤＬ)对牛胸主动脉血管平滑肌细胞(ＳＭＣｓ)ＤＮＡ合成及细胞增殖的影响 ,探讨了血脂康抗ＡＳ可能的作用机制。　　一、材料和方法　　 1.试剂和仪器 :胸腺嘧啶核苷酸 (3 Ｈ ｔｈｙｍｉｄｉｎｅ ,3 Ｈ Ｔｄ) :上海原子核研究所产品 (1ｍＣｉ/ｍｌ ,1ｍＣｉ=37ＭＢｑ) ;小牛血清 :杭州四季清生物制品公司产品 ;血脂康原粉 :北大维信科技有限公司产品〔…     

泽泻汤对氧化型低密度脂蛋白诱导血管平滑肌细胞增殖的影响

目的观察泽泻汤对血管平滑肌细胞(VSMC)周期蛋白Cyclin D1、Cyclin E、增殖细胞核抗原(PCNA)和p27表达的影响,探讨泽泻汤在氧化型低密度脂蛋白(ox-LDL)诱导的VSMC增殖中的作用和机制。方法通过体外50 mg/L ox-LDL诱导VSMC,建立VSMC增殖的动脉粥样硬化细胞模型;应用空白血清及20%泽泻汤含药血清干预。MTS法检测泽泻汤含药血清对VSMC增殖的影响;Western blot检测增殖相关蛋白Cyclin D1、Cyclin E、PCNA和p27表达水平。结果 50 mg/L ox-LDL可明显诱导VSMC增殖。与ox-LDL组比较,泽泻汤含药血清可显著抑制ox-LDL诱导的VSMC增殖,下调细胞Cyclin D1、Cyclin E和PCNA的表达,同时促进p27蛋白表达。结论泽泻汤具有抗VSMC增殖的作用,机制可能与上调p27蛋白和抑制Cyclin D1、Cyclin E、PCNA表达有关。     

氧化低密度脂蛋白对血管内皮细胞骨架的损伤及其机制

目的:观察氧化低密度脂蛋白(OxLDL)对血管内皮细胞(ECV-304)微丝肌动蛋白的影响及同细胞内钙离子变化间的关系,以探讨OxLDL的致动脉粥样硬化机制。方法:采用ECV 304内皮细胞株体外培养,分为空白对照组,OxLDL (200 μg/ml)组,采用激光扫描共聚显微镜分别观察OxLDL作用后12小时胞内钙离子的变化及OxLDL作用24小时后的细胞骨架微丝(F-actin)的改变。结果:OxLDL作用12小时后细胞内钙离子浓度明显升高(P<0.001),而F-actin也在24小后发生明显的破坏。结论:OxLDL可破坏内皮细胞的骨架结构从而导致血管内皮细胞的屏障功能的损害,这一作用可能与动脉粥样硬化形成有关。     

血管内皮细胞脂质过氧化损伤对平滑肌细胞增殖的影响

血管内皮细胞脂质过氧化损伤对平滑肌细胞增殖的影响高岩，陈铁镇，杨向红（中国医科大学实验病理学研究室，沈阳１１０００１）近年来，脂质过氧化作用（ＬＰＯ）与动脉粥样硬化（Ａｓ）的关系很受重视。本研究室以往的实验已证实，脂质过氧化作用不仅可引起培养人血管内...     

脑心通对氧化低密度脂蛋白诱导血管平滑肌细胞增殖及凋亡的影响

血管平滑肌细胞(VSMC)的异常增殖是动脉粥样硬化(AS)形成的关键因素.研究表明,氧化型低密度脂蛋白(oxLDL)可以刺激VSMC的增殖,促进AS的发生和发展.应用药物抑制VSMC增殖可以有效地减少AS的发生.     

人血管内皮细胞氧化低密度脂蛋白基因表达谱分析

目的 检测内皮细胞中与低密度脂蛋白氧化可能相关的信号传导相关蛋白基因,期望获得阐明氧化机制的有价值的线索.方法 利用低密度脂蛋白作用人血管内皮细胞系ECV304,通过基因芯片分析人信号传导相关蛋白的表达谱.结果 在被检测的1 990个基因中,52个基因的表达变化超过了2倍以上,其中23个基因表达增加,29个基因表达减少.在表达发生改变的基因中,有7个已在最新的报道中认为可能和动脉粥样硬化相关.结论 我们的发现可能给内皮细胞氧化低密度脂蛋白的机制的揭示提供全新的思路.     

Hyperlipemic-very low density lipoprotein, intermediate density lipoprotein and low density lipoprotein act synergistically with serotonin on vascular smooth muscle cell proliferation

BACKGROUND: Previous studies have shown that very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) and low density lipoprotein (LDL) from hyperlipidemic plasma are more atherogenic than those from normal plasma. Since platelet aggregation at sites of atherosclerotic injury exposes the cells to high concentrations of serotonin (5HT), a known mitogen for vascular smooth muscle cells (VSMCs), it was examined whether VLDL, IDL or LDL from plasma of 1% cholesterol-fed rabbits can potentiate the mitogenic effect of 5HT on VSMC. METHODS: Growth arrested primary aortic VSMC in 1st or 2nd passage were incubated with different concentrations of VLDL, IDL or LDL in the presence or absence of pertusis toxin (PTX) for 24 h followed by incubation with 5HT for 24 h. The amount of [3H]thymidine incorporated into the DNA as well as the increase in cell number was measured. RESULTS: Either VLDL, IDL or LDL at a concentration of 60 microg/ml induced proliferation of VSMC by themselves (196, 137 or 122% increase in [3H]thymidine incorporation, or 122, 119 or 122% increase in cell number, respectively when compared to the control, P<0.05). This effect on DNA synthesis was markedly potentiated by 50 microM 5HT to 465, 714 and 1369%, respectively. PTX reversed the mitogenic effect of 5HT, but not that of VLDL, IDL or LDL. Conclusion: These results suggest that even low concentration of VLDL, IDL or LDL from hypercholesterolemic plasma may significantly potentiate the mitogenic effect of 5HT, that is released by aggregating platelets at sites of vascular damage.     

Effect of glycated low density lipoprotein on smooth muscle cell proliferation.

BACKGROUND: We investigated the effect of glycated low density lipoprotein (LDL) on smooth muscle cell proliferation. METHODS: Blood was drawn from 6 healthy subjects after overnight fasting. Native LDL was obtained by separating LDL from the samples with sequential ultracentrifugation. Glycated LDL was prepared by glycating the native LDL in vitro. Native and glycated LDL were added to a medium containing cultured porcine coronary artery smooth muscle cells, and the change in cell proliferation was examined after 24, 48, 72, and 96 hs. The cells were counted using a cell counting kit (Dojin Chemical Co., Ltd.). RESULTS: There was no significant difference in the cell count between the control group, in which only PBS was added, and the native LDL group. However, cell proliferation was appreciably higher in the glycated LDL group than in the native LDL group. The mean total cell count at 24, 48, 72 and 96 hs was significantly higher (p<0.01) in the glycated LDL group (median: 0.843; range: 0.576-1.060) than in the native LDL group (median: 0.541; range: 0.282-0.683). CONCLUSIONS: These findings suggest that glycated LDL induces significantly greater acceleration of smooth muscle cell proliferation than does native LDL. Therefore, the acceleration of smooth muscle cell proliferation requires modification of LDL.     

氧化修饰低密度脂蛋白对大鼠血管平滑肌细胞基质金属蛋白酶—2和骨桥蛋白基因表达及细胞增殖的影响

目的 探讨氧化修饰低密度脂蛋白（oxLDL)和天然低密度脂蛋白（nLDL)促血管平滑肌细胞（VSMC）增殖是否与细胞外基质（ECM）降解及粘附蛋白合成有关。方法 应用Northern印迹和^3H-TdR掺入的方法,观察两种脂蛋白对VSMC表达基质金属蛋白酶－2（MMP－2）和骨桥蛋白（OPN）的影响,以及ECM代谢与VSMC增殖之间的关系。结果 10μg/ml oxLDL作用于细胞24h可使MMP－2和OPN基因表达活性增加1倍左右;nLDL对两种基因表达的诱导作用较弱,高浓度的oxLDL对MMP－2和OPN表达不产生明显的影响。在一定浓度和时间范围内,LDL促细胞增殖作用显示量－效与时－效依赖关系。结论 oxLDL和nLDL可在多位点上发挥促进VSMC增殖和迁移的作用。     

氧化型低密度脂蛋白对人血管平滑肌细胞透明质酸表达的影响及机制

目的观察氧化型低密度脂蛋白(ox-LDL)对人主动脉血管平滑肌细胞透明质酸(HA)合成的影响,并初步探究其分子机制。方法体外培养人主动脉血管平滑肌细胞T/G HAVSMC,使用不同浓度(10、25、50、75、100 mg/L)ox-LDL对T/G HAVSMC细胞进行干预,使用CCK-8法检测细胞存活率,并进行分组分析。采用浓度为25和50 mg/L的ox-LDL干预T/G HAVSMC细胞48 h,并设置天然LDL(native-LDL,N-LDL)组(50 mg/L的N-LDL)和对照组,使用HPLC法测定HA含量,使用实时定量PCR检测透明质酸合成酶2(HAS2)和透明质酸合成酶3(HAS3)的mRNA表达,使用Western blot法检测凝集素样氧化型低密度脂蛋白受体1(LOX-1)、低密度脂蛋白受体相关蛋白1(LRP-1)、氧化脂蛋白的清道夫受体(SR-PSOX)以及脂肪酸转位酶(CD36)的表达。结果低于100 mg/L的ox-LDL对T/G HAVSMC细胞无明显细胞毒性。25 mg/L和50 mg/L ox-LDL组HA含量、HAS2和HAS3的mRNA表达量明显高于N-LDL组和对照组(P0.05),N-LDL组与对照组组间差异无显著性(P0.05)。25 mg/L和50 mg/L ox-LDL组LOX-1表达明显高于N-LDL组和对照组(P0.05),而LRP-1、SR-PSOX和CD36表达无明显变化(P0.05)。结论 ox-LDL能够诱导人主动脉平滑肌细胞中HA的合成,其机制可能与结合LOX-1有关。     

Impact of oxidized low density lipoprotein on vascular cells

Oxidized LDL (OxLDL) is a proatherogenic lipoprotein, accumulating in the vascular wall and contributing to the pathogenesis of vascular dysfunction early in the development of atherosclerosis. Enhanced serum levels of OxLDL, as well as antibodies against its epitopes, are predictive for endothelial dysfunction and coronary heart disease. While enhanced oxidative stress is one factor triggering formation of OxLDL, OxLDL itself has been identified as a potent stimulus for vascular oxygen radical formation, causing a vicious circle. OxLDL-induced O(2)(-) formation, largely through activation of NADPH oxidase, but also through uncoupling of endothelial NO-synthase and through direct O(2)(-) release, leads to endothelial dysfunction. Furthermore, OxLDL-induced O(2)(-) formation has a strong impact on tissue remodeling, resulting in either cell growth - proliferation or hyperplasia - or apoptotic cell death. The effect of OxLDL on cell cycle regulation is mediated by activation of the small GTPase RhoA and consequent regulation of p27(KIP1), a key enzyme of the cell cycle. In addition, OxLDL-induced activation of RhoA sensitizes the contractile apparatus of the vessel wall, enhancing the contractile tonus and favoring vasospasm. Thus, through a variety of mechanisms, OxLDL importantly contributes to vascular dysfunction and remodeling.     

Mildly oxidized low-density lipoprotein acts synergistically with angiotensin II in inducing vascular smooth muscle cell proliferation

OBJECTIVES: Considerable attention has been focused on both mildly oxidized low-density lipoprotein (mox-LDL) and highly oxidized LDL (ox-LDL) as important risk factors for cardiovascular disease. Further, angiotensin II (Ang II) appears to play a crucial role in the development of hypertension and atherosclerosis. We assessed the effect of oxidatively modified LDL and its major oxidative components, i.e., hydrogen peroxide (H2O2), lysophosphatidylcholine (LPC), and 4-hydroxy-2-nonenal (HNE) and their interaction with Ang II on vascular smooth muscle cell (VSMC) DNA synthesis. METHODS: Growth-arrested rabbit VSMCs were incubated in serum-free medium with different concentrations of native LDL, mox-LDL, ox-LDL, H2O2, LPC, or HNE with or without Ang II. DNA synthesis in VSMCs was measured by [3H]thymidine incorporation. RESULTS: Ang II stimulated DNA synthesis in a dose-dependent manner with a maximal effect at a concentration of 1 micromol/l (173%). Ang II (0.5 micromol/l) amplified the effect of native LDL at 500 ng/ml, ox-LDL at 100 ng/ml, and mox-LDL at 50 ng/ml on DNA synthesis (108 to 234%, 124 to 399%, 129 to 433%, respectively). H2O2 had a maximal effect at a concentration of 5 micromol/l (177%), LPC at 15 micromol/l (156%), and HNE at 0.5 micromol/l (137%). Low concentrations of H2O2 (1 micromol/l), LPC (5 micromol/l), or HNE (0.1 micromol/l) also acted synergisitically with Ang II (0.5 micromol/l) in inducing DNA synthesis to 308, 304, or 238%, respectively. Synergistic interactions of Ang II (0.5 micromol/l) with mox-LDL, ox-LDL (both 50 ng/ml), H2O2 (1 micromol/l), LPC (5 micromol/l), or HNE (0.1 micromol/l) on DNA synthesis were completely reversed by the combined use of probucol (10 micromol/l), a potent antioxidant and candesartan (0.1 micromol/l), an AT1 receptor antagonist. CONCLUSIONS: Our results suggest that mox-LDL, ox-LDL, and their major components H2O2, LPC, and HNE act synergistically with Ang II in inducing VSMC DNA synthesis. A combination of antioxidants with AT1 receptor blockade may be effective in the treatment of VSMC proliferative disorders associated with hypertension and atherosclerosis.     

辛伐他汀对氧化型低密度脂蛋白诱导的猪冠状动脉平滑肌细胞增殖和迁移的影响

目的观察辛伐他汀对氧化型低密度脂蛋白(OX-LDL)诱导的猪冠状动脉平滑肌细胞(SMC)增殖和迁移的影响。方法采用体外猪冠状动脉SMC培养技术,以3H-TdR的参入量表示冠状动脉的SMCDNA合成情况,应用划线方法测定冠状动脉SMC的迁移距离,观察辛伐他汀对OX-LDL诱导的冠状动脉SMC增殖和迁移的影响。结果浓度为10-8~10-5mol/L的辛伐他汀可使猪冠状动脉SMC的3H-TdR参入量减少,与对照组相比差异具有统计学意义;且药物浓度越高,参入量越少。10-8~10-5mol/L辛伐他汀均可使猪冠状动脉SMC的迁移距离减少,与对照组相比差异具有统计学意义。结论辛伐他汀可剂量依赖性地抑制OX-LDL诱导的猪冠状动脉SMC的增殖和迁移。     

正五聚素蛋白3在氧化型低密度脂蛋白诱导的平滑肌细胞中的表达

目的通过体内和体外实验探讨正五聚素蛋白3(pentraxin 3,PTX3)与动脉粥样硬化的关系,以及在氧化型低密度脂蛋白(ox-LDL)诱导的平滑肌细胞增殖和迁移中的作用。方法选择雄性载脂蛋白E基因敲除小鼠20只,随机分为高脂饮食组和正常饮食组,每组10只。饲养16周后行主动脉超声检查,处死后取主动脉行苏木精-伊红染色及PTX3免疫组织化学测定。小鼠主动脉平滑肌细胞传代培养后,分别与不同浓度ox-LDL(0、20、50、100mg/L)共同孵育,选增殖最明显100mg/L ox-LDL作为实验组,并以正常主动脉平滑肌细胞作为对照组。检测平滑肌细胞增殖、迁移程度,PTX3蛋白表达水平。结果高脂饮食组动脉粥样硬化斑块程度较正常饮食组严重[(29.71±2.07)%vs(3.26±0.81)%,P0.01]。实验组主动脉平滑肌细胞增殖、迁移和PTX3表达较对照组明显增高(P0.05,P0.01)。结论 PTX3参与了动脉粥样硬化的形成,并与脂质代谢相关。