血管平滑肌细胞迁移、增殖致冠状动脉搭桥术后再狭窄分子机制的研究进展

血管平滑肌细胞（Vascular Smooth Muscle Cell，VSMC）增殖和凋亡涉及冠状动脉搭桥（Coronary Artery Bypass Graft，CABG）术后损伤血管异常再塑的病原学和再狭窄的发病机制。CABG术后血管新生内膜形成和管壁重构再塑过程中，血管内皮细胞（Vascular Endothelial Cell，VEC）的损伤导致多种炎症因子和生长调节因子的表达、激活紊乱，诱导VSMC进行表型调变（Made Letion），使其对损伤后的细胞增殖和凋亡调节失常；VSMC细胞周期调节因子、凋亡程序性基因和细胞外基质（Extra Cellular Matrix，EcM）的表达异常，刺激VSMC从血管中层向内膜迁移．发生过度增殖和凋亡抑制，并导致ECM合成增加和脂质沉积，引起管壁纤维化和粥样硬化病变的发生和进展，是CABG术后再狭窄的重要发病机制。     

microRNAs调控血管平滑肌细胞增殖和迁移的研究进展

血管平滑肌细胞(VSMC)增殖和迁移是动脉粥样硬化和血管成形术后再狭窄发生、发展的重要原因,近年研究发现微核糖核酸(miRs)通过调节一些转录因子的表达在VSMC的增殖和迁移过程中发挥着重要的作用;miRs是一类约由22个核苷酸组成的内源性非编码小分子RNAs,主要通过抑制转录后基因表达或促进靶基因m RNAs降解发挥调节作用,研究发现多种miRs在VSMC中特异性地表达,有可能成为血管疾病新的生物标志物和治疗靶点。文章就miR-21、miR-26a、miR-130a、miR-133、miR-143/145、miR-146a、miR-221/222和miR-663等多种miRs通过调节一些转录因子的表达在VSMC的增殖和迁移过程中的作用机制分别进行了综述。     

miR-145对血管平滑肌细胞生物活性及SM22α mRNA表达的作用*

目的：探究miR-145 对血管平滑肌细胞（VSMC）的生物活性及对SM22α mRNA 表达的作用。方法： VSMC分为3 组,增殖VSMC组（ 增殖组）、增殖VSMC + 转染miR-145-NC 组（ 空载体组）、增殖VSMC+转染 miR-145 组（ 增殖转染组）。采用RT-PCR 检测VSMC中SM22α mRNA、miR-145 的表达;免疫印迹检测cyclin E、p27 蛋白水平;Transwell 小室检测VSMC侵袭能力;MTT检测VSMC活力;流式细胞术检测VSMC凋亡率。 结果：增殖组VSMC中SM22α mRNA、miR-145 水平与空载体组相比数值较为接近;与增殖组及空载体组相比, 增殖转染组VSMC中miR-145、SM22α mRNA 表达升高。增殖组VSMC中cyclin E、p27 蛋白水平与空载体组相 比无差异;增殖转染组cyclin E 蛋白水平低于空载体组、增殖组,p27 蛋白水平高于空载体组、增殖组。增殖组 VSMC迁移数量与空载体组相比无差异;增殖转染组VSMC迁移数量明显低于空载体组、增殖组。增殖组与空载 体组VSMC在不同时间点的OD 值均较为接近;增殖转染组OD 值在不同时间点均低于空载体组、增殖组。增殖 组VSMC凋亡率与空载体组数值接近,组间比较差距较小;增殖转染组VSMC凋亡率较空载体组和增殖组升高。 结论：过表达miR-145 通过促进SM22α 水平增加,抑制血管平滑肌细胞的增殖,并促进其凋亡。     

脂质体介导的VEGF基因对培养血管细胞增殖的影响

目的：观察脂质体介导转移的VEGF基因在血管平滑肌细胞（VSMC）中的表达，比较VEG对血管内皮细胞（VEC）及VSMC增殖的影响。方法：将含血管内皮生长因子（VECF）CDNA的真核表达载体pSV121用脂质体介导的基因转移法导入血管平滑肌细胞（VSMC）中，取此VSMC条件培养液，再行血管内皮细胞（VEC）培养，用Northmblot杂交，Westem印迹法及[~3H]胸腺嘧啶核苷掺入法，观察了VEGF在VSMC及其条件培养液中的表达，比较了VEGF对VEC及VSMC增殖的影响。结果：转基因组VSMC中VEGFmRNA呈稳定高表达，转基因组VSMC条件培养液中，VEGF抗原表达显著高于对照组（P均＜0．01），加入此条件培养液的各组VEC的[~3H]胸腺嘧啶核苷掺入量均显著高于对照组（p均＜0．01），而在VSMC组无显著差异。结论：VEGF的转录和表达表明脂质体介导的血管细胞VEGF基因转移是成功的。VEGF具有促VEC增殖作用，但对VSMC无促增殖作用，有利于缺血性疾病及血管再狭窄的防治。     

γ射线辐射对大鼠胸主动脉平滑肌细胞肾上腺髓质素和内皮素生成的影响

为观察^60COγ射线辐射对培养的大鼠血管平滑肌细胞（VSMC）肾上腺髓质素（Adm)内皮素（ET）生成的影响，以探讨辐射防治再狭窄的机制，采用逆转录－竞争性聚合酶链反应观察经0,1,,24,25Gy辐射的培养大鼠血管平滑肌细胞Adm和ET mRNA水平；用放免方法测定相应的Adm和ET的生成。发现1Gy辐射对VSMC Adm和ET的生成及基因表达均无显著影响。14Gy辐射后VSMC生成的Adm较对照组增加270％（P＜0．05），ET的生成较对照组降低27．3％（P＜0．01）；25Gy辐射使VSMC生成的Adm和ET含量分别增加233％（P＜0．05）和降低58．0％（P＜0．01）。14和25Gy辐射分别使细胞Adm mRNA水平较对照组增加82．4％（P＜0．01）和101％（P＜0．01），使ET mRNA水平较对照组降低47．1％（P＜0．01）及40．2％（P＜0．01）。表明^60Coγ射线辐射可以促进Adm的蛋白及基因表达，而抑制ET的蛋白及基因表达，增加了Adm/ET比值。     

移植静脉平滑肌增殖与调节的研究

移植静脉术后狭窄是影响远期通畅率的重要问题。术中造成血管壁损伤所致的平滑肌细胞迁移、增殖引起内膜增生是狭窄的重要原因之一 〔1〕。受损部位多种细胞所释放的生长因子、细胞因子和机械因素 ,在血管平滑肌细胞 (VSMC)增殖的调节中 ,起着重要作用 ,现综述如下。1　内皮素 (ET)ET是 Yanagisawa等于 1988年从猪主动脉内皮细胞培养液中分离获得的 ,是目前发现的作用最强 ,持续最久的缩血管多肽。在生理状态下合成、释放较少 ,作用于平滑肌 ,其机制还不是很清楚。 ET有 4种以上亚型家族 :ET- 1、 ET- 2、ET- 3和血管活性肠收缩肽等…     

C反应蛋白促进离体SHR大鼠血管平滑肌细胞的增殖

目的： 探讨C反应蛋白（CRP）对SHR大鼠主动脉平滑肌细胞（VSMC）增殖的影响。方法： SHR大鼠VSMC培养，取3-4代细胞进行药物干预。先加入CRP和CRP受体CD32的抗体，测定NF-κB和抑制蛋白I-κB在VSMC中的表达；再使用NF-κB活化抑制剂PDTC，观察对血管紧张素Ⅱ诱发VSMC增殖的影响。结果： 使用CRP后，SHR大鼠VSMC的NF-κB和抑制蛋白I-κB的表达增强；使用CD32抗体可以抑制这种增强； PDTC可以抑制血管紧张素Ⅱ促进平滑肌细胞的增殖作用。结论： CRP可以通过直接激活SHR大鼠VSMC的 NF-κB，促进其VSMC增殖。     

肾上腺髓质素对血管紧张素II的促血管平滑肌细胞增殖作用的影响

观察肾上腺髓质素前体N端20肽（PAMP）和肾上腺髓质素（ADM ）对血管紧张素II（AngII）诱导的血管平滑肌细胞（VSMC）增殖的影响。用测定培养的大鼠VSMC3H-TdR掺入和蛋白激酶C（PKC）活性的方法。结果发现：10-8mol/L AngII刺激培养的VSMCs 3H-TdR掺入增加2.68倍（P＜0.01）、PKC活性增加1.02倍（P＜0.01）。而10-9mol/L-10-7mol/L的PAMP或ADM与10-8mol/L AngII同时孵育，则显著抑制AngII的上述作用（P＜0.01）。且两者的抑制作用无明显差异。PAMP和ADM均有抑制AngII所致的VSMC增殖作用，可能是通过抑制信号传导系统中的PKC活性来实现的。PAMP和ADM 与AngII相互作用，共同调节VSMC的增殖，从而维持循环系统功能的稳态。     

半边莲生物碱抑制内皮素-1诱导的人脐动脉平滑肌细胞增殖

目的：探讨半边莲生物碱(LCLA)对内皮素-1（ET-1）诱导人脐动脉平滑肌细胞（VSMC）增殖的抑制效应及其作用机制。 方法： 采用细胞计数、［3H］-TdR掺入、流式细胞术、免疫细胞化学染色及Fura-3/AM 荧光探针标记方法检测VSMC增殖；并用台盼蓝拒染、乳酸脱氢酶（LDH）检测方法观察LCLA对VSMC的毒性反应。 结果： LCLA(100、200和400 mg/L)、BQ-123(10-6mol/L)、ST(10-7mol/L)均抑制ET-1所诱导的VSMC增殖（均P＜0.05）：明显降低VSMC的细胞数目、［3H］-TdR掺入量、S期和G2/M期的数目百分比，增加G0/G1期的数目百分比；减弱细胞内增殖细胞核抗原（PCNA）的表达强度和Ca2+荧光强度；LCLA的抑制作用存在明显的剂量依赖关系，但对VSMC存活率和LDH释放量均没有影响。 结论： LCLA浓度依赖性地抑制ET-1所诱导的人脐动脉VSMC增殖，其机制与降低VSMC内Ca2+含量有关。     

原花青素对同型半胱氨酸诱导的血管平滑肌细胞增殖、迁移的影响

目的： 探讨葡萄籽中的原花青素(GSP)对同型半胱氨酸(HCY)诱导的血管平滑肌细胞(VSMC)增殖、迁移的抑制作用及其机制。方法: 采用细胞计数及［3H］-TdR检测细胞增殖，用DCFH-DA、Western blotting法检测GSP对VSMC增殖、迁移的抑制作用及分析其相关的分子信号转导通路。结果: HCY浓度依赖性地诱导VSMC的增殖与迁移, 1 mmol/L HCY使VSMC的增殖与迁移分别比对照组升高3.9倍及4.1倍(P<0.01)；DCFH-DA结果显示, 1 mmol/L HCY使VSMC的活性氧(ROS)水平比对照组升高4倍(P<0.01)。而在不同浓度的GSP(5-20 g/L) 处理组，HCY诱导VSMC的增殖、迁移以及活性氧水平都被显著抑制(P<0.01)。在GSP 20 g/L 处理组，VSMC的增殖、迁移以及活性氧水平都接近空白对照组。与HCY组相比，GSP还显著降低HCY诱导VSMC MCP-1、IL-6及TNF-α的表达水平(P<0.01)。GSP还阻断HCY诱导的NF-κB的激活及显著降低NF-κB的活性(P<0.01)。结论: GSP通过抑制NF-κB的激活而抑制HCY诱导的血管平滑肌细胞增殖、迁移及炎症反应。     

Corresponding distributions of increased endothelin-B receptor expression and increased endothelin-1 expression in the aorta of apolipoprotein E-deficient mice with advanced atherosclerosis

Endothelin (ET)-1 causes proliferation of vascular smooth muscle cells (VSMC). Although it has been reported that stimulation of ET(B) receptors as well as ET(A) receptors promote proliferation of VSMC, the precise distribution of each receptor subtype in atherosclerotic vessels is unknown. Previous studies demonstrated that apolipoprotein E (apoE)-deficient mice have hypercholesterolaemia and develop severe atherosclerosis. To investigate the pathophysiological roles of vascular ET system in atherosclerosis, we examined preproET-1 messenger ribonucleic acid expression in the aorta of apoE-deficient mice, and performed immunohistochemical staining for ET-1 and each ET receptor subtype (ET(A) and ET(B) receptors) in the atherosclerotic lesions of these mice. The level of preproET-1 mRNA in the aorta was significantly higher in the apoE-deficient mice than in the control mice. Strong ET-1 staining was observed in the macrophage-foam cells, intimal and medial VSMC in the atherosclerotic lesions of the apoE-deficient mice. In addition, in the atherosclerotic lesions, strong ET(B) receptor staining was observed in the macrophage-foam cells, intimal and medial VSMC, which distribution corresponded closely to that of ET-1. ET(A) receptor staining was observed in the medial VSMC of both groups, but not in the macrophage-foam cells of the apoE-deficient mice. ET(A) receptor staining in the medial VSMC was stronger in the apoE-deficient mice than in the control mice. These results suggest that the vascular ET system, including ET-1 and ET receptors, is activated in the atherosclerotic lesions of apoE-deficient mice. Since the distribution of strong ET(B) receptor staining corresponded closely to that of ET-1, it is suggested that the ET system, mediated by ET(B) receptors, has an important role in the pathophysiology of the atherosclerotic lesions of apoE-deficient mice.     

纤维蛋白原对人血管平滑肌细胞株增殖和移行的影响

目的:探讨纤维蛋白原(Fg)、纤维蛋白(Fb)及其降解产物(FDPs)对体外人血管平滑肌细胞(VSMC)增殖与移行的影响。方法:采用细胞计数法和四唑盐(MTT)比色法测定VSMC增殖,通过刮伤实验(woundingmodel)和Transwell嵌套装置观察VSMC的移行。结果:Fg本身对VSMC增殖无促进作用,但可促进VSMC的移行,且呈浓度依赖关系;Fb及FDPs均可明显促进VSMC增殖和移行,Fb的作用还呈现浓度依赖关系,而FDPs作用呈现先增强后减弱的趋势,即在一定浓度范围内作用明显,高于或低于此范围作用较弱。结论:Fb及FDPs通过促VSMC的增殖和移行作用,可能在再狭窄和动脉粥样硬化中起重要作用。     

The role of vascular smooth muscle cells on the pathogenesis of atherosclerosis

Atherosclerosis is the leading cause of death and disability. The lesions of atherosclerosis represent a series of highly specific cellular and molecular responses. The earliest changes that precede the formation of lesions of atherosclerosis take place in the endothelium (EC), with resultant endothelial dysfunction. The EC-induced injury can result in increased lipid permeability, macrophage recruitment, formation of foam cells, and recruitment of T-lymphocytes and platelet. After intimal injury, different cell types,including ECs, platelets, and inflammatory cells release mediators, such as growth factors and cytokines that induce multiple effects including phenotype change of vascular smooth muscle cells (VSMC) from the quiescent "contractile" phenotype state to the active "synthetic" state, that can migrate and proliferate from media to the intima. The inflammatory response simulates migration and proliferation of VSMC that become intermixed with the area of inflammation to form an intermediate lesion. These responses continue uninhibited and is accompanied by accumulation of new extra cellular matrix (ECM). The migratory and proliferative activities of VSMC are regulated by growth promoters such as platelet derived growth factors (PGF), endothelin-1 (ET-1), thrombin, fibroblast growth factor (FGF), interleukin-1 (IL-1) and inhibitors such as, heparin sulfates , nitric oxide (NO), transforming growth factor (TGF)-beta. The matrix metallo proteinases (MMPs) could also participate in the process of VSMC migration. MMPs could catalyze and remove the basement membrane around VSMC and facilitate contacts with the interstitial matrix. This could promote a change from quiescent, contractile VSMC to cells capable of migrating and proliferating to mediate repair. The VSMC regulation is a very complex process, VSMC are stimulated to proliferate and migrate by some kind of cytokines, growth factors, angiotensin II (Ang-II). Together with apoptosis, proliferation and migration of VSMC are vital to the pathogenesis of atherosclerosis and plaque rupture. Rupture of the plaque is associated with increased fibrous cap macrophage, increased VSMC apoptosis, and reduced fibrous cap VSMC. VSMC are the only cells with plaques capable of synthesizing structurally important collagen isoforms, and the apoptosis of VSMC might promote plaque rupture.     

Stimulation of vascular smooth muscle cell migration by macrophage migration inhibitory factor

Macrophage migration inhibitory factor (MIF) is a well known proinflammatory factor that influences the migration and proliferation of various cell types, predominantly monocytes and macrophages. Recent evidence suggests an important role for MIF in the progression of atherosclerosis and restenosis. For this reason, we studied the effect of MIF on platelet-derived growth factor-BB (PDGF-BB)-induced migration and PDGF receptor protein expression in vascular smooth muscle cells (VSMCs). Furthermore, the possibility of MIF influencing the migration of VSMCs was investigated. Our results show that short-term incubation of MIF is able to enhance PDGF-BB-induced migration. Long-term incubation decreases PDGF-BB-induced migration, but preserves a short-term stimulatory effect. These effects are not regulated at the level of PDGF receptor protein expression. MIF also acts as a chemoattractant for VSMCs, with a maximum response at 15 ng/ml. In contrast, the proliferation of VSMCs was unaffected by MIF. We conclude that MIF has a biphasic effect on VSMC migration. It remains unclear whether this effect is direct or involves the secretion of unidentified promigratory factors. Exogenous MIF does not stimulate VSMC proliferation; however, a role for MIF in proliferation cannot be fully ruled out. In view of the known key contributions of macrophage-derived MIF and VSMCs, the observed effects may well play a role in the progression of atherosclerosis and restenosis.     

Oxidative Stress Produced with Cell Migration Increases Synthetic Phenotype of Vascular Smooth Muscle Cells

Phenotypic modulation of vascular smooth muscle cells (VSMC) and reactive oxygen species (ROS) is important in vascular pathogenesis. Understanding how these factors relate to cell migration can improve design of therapeutic interventions to control vascular disease. We compared the proliferation, protein content and migration of cultured aortic VSMC from wild type (WT) versus transgenic mice (Tg^p22phox), in which overexpression of p22phox was targeted to VSMC. Also, we compared H₂O₂ generation and expression of specific phenotypic markers of non-migrating with migrating WT versus Tg^p22phox VSMC in an in vitro wound scratch model. Enhanced H₂O₂ production in Tg^p22phox versus WT VSMC (p < 0.005) significantly correlated with increased protein content, proliferation, and migration. VSMC migrating across the wound edge produced more H₂O₂ than non-migrating VSMC (p < 0.05). The expression of synthetic phenotypic markers, tropomyosin 4 and myosin heavy chain embryonic (SMemb), was enhanced significantly, while the expression of contractile marker, smooth muscle α-actin, was reduced significantly in migrating versus non-migrating cells, and also in Tg^p22phox versus WT (p < 0.005) VSMC. These results are consistent with increased production of ROS accelerating the switch from the contractile to the synthetic phenotype, characterized by increases in proliferation, migration, and expression of TM4 and SMemb and decreased α-actin.     

β3整合素在生长因子促血管平滑肌细胞粘附和迁移中的作用

目的:探讨血小板衍生生长因子(PDGF)对β₃整合素表达的影响及β₃整合素在PDGF促血管平滑肌细胞(VSMC)粘附、迁移和增殖中的作用。方法:用抗β₃整合素胞外域抗体封闭VSMCβ₃整合素后,通过-TdR掺入、细胞粘附、细胞迁移分析及RT-PCR等方法,检测PDGF对β₃整合素表达及对VSMC粘附、迁移和增殖的影响。结果:阻断β₃整合素与细胞外基质(ECM)蛋白相互作用后,可在一定程度上抑制PDGF刺激的VSMC增殖,使-TdR掺入率降低39%;在β₃整合素抗体浓度为10mg/L时,PDGF引发的细胞粘附和迁移受到显著抑制(P＜0.05);PDGF作用于VSMC6h,β₃整合素基因表达活性达到峰值,比对照细胞高87%。结论:PDGF可显著诱导β₃整合素在VSMC中表达;β₃整合素与ECM蛋白相互作用在PDGF促VSMC粘附、迁移方面发挥重要作用。