血红素加氧酶-1在对抗过氧化氢引起的血管低反应性中的作用

目的：研究HO-1的诱导剂是否可对抗H2O2引起的血管低反应性,并探讨其作用机制。方法：采用血管环灌流装置,观察胸主动脉环的收缩效应。结果：①SD大鼠腹腔注射高铁血红素后,主动脉HO-1活性和血中CO含量增高;同时,H2O2引起的血管收缩功能下降的现象明显改善。②KATP通道阻断剂优降糖,而非GC抑制亚甲蓝,可取消高铁血红素的抗H2O2损伤的作用。③Hemin＋H2O2组与单纯H2O2组的钙收缩曲线无明显差异。④无钙液中,高铁血红素可抑制H2O2引起的咖啡因和PE诱导的收缩幅度的下降。结论：诱导主动脉HO-1活性增加,可对抗氧化应激引起的血管收缩反应的低下,其机制可能是通过激活KATP通道,影响细胞内贮存钙的释放起作用。而与GC信号转导通路无关。     

血红素加氧酶-1在缺血/再灌注损伤中的保护作用

血红素加氧酶-1(Heme Oxygenase-1,HO-1)是催化血红素分解的关键酶。近年来,人们对血红素降解产物的抗氧化、抗炎症等功能的认识推动了对HO酶系的研究。缺血／再灌注损伤(IRI)是一个重要的临床问题,而临床上对IRI的防治尚缺乏有效的方法。目前发现HO-1过表达具有抗IRI的作用,其保护作用的可能机制有：抗氧化作用、调节微循环、调节细胞周期和抗炎症作用。     

人SOD1基因在大鼠血管平滑肌细胞的表达及对抗氧自由基损伤的作用

本实验构建含人铜锌超氧化物歧化酶（ｈＳＯＤ１）基因的逆转录病毒载体,将其导入离体培养的鼠血管平滑肌细胞,观察ｈＳＯＤ１基因表达及其抗氧自由基损害作用,结果表明：（１）载体构建策略和方法正确,ｈＳＯＤ１基因可在靶细胞中高效稳定表达;（２）转化ｈＳＯＤ１的ＶＳＭＣｓ可对抗大剂量氧自由基对细胞的直接损伤作用;（３）小剂量氧自由基刺激ＶＳＭＣｓ增殖,而转化ｈＳＯＤ１的ＶＳＭＣｓ增殖反应受到抑制,本研究结果     

逆转录病毒介导诱导型一氧化氮合酶基因转染抑制血管平滑肌细胞增殖的研究

目的：研究逆转录病毒介导诱导型一氧化氮合酶（iNOS）基因转染对体外培养的大鼠主动脉血管平滑肌细胞（VSMC）增殖的影响,探讨iNOS转基因治疗血管移植术后再狭窄的可行性。方法：将不同滴度的病毒上清转染体外培养的VSMC;采用RT-PCR、Western-blot检测VSMC内iNOSmRNA和iNOS蛋白的表达;用Griess法检测iNOS转基因细胞的培养液中一氧化氮（NO）的含量;用改良MTT、法检测iNOS转基因对VSMC增殖的抑制作用。结果：不同滴度的PLXSNiNOS转染体外培养的VSMC48h后,在VSMC内可检测到外源性iNOSmRNA和iNOS蛋白,表达水平随病毒滴度的增加而增强,呈现剂量依赖性;而用最高滴度的PIXSN转染体外培养的VSMC48h后,在VSMC内未能检测到外源性iNOSmRNA和iNOS蛋白表达;iNOS转基因细胞的培养液中NO含量显著增高,同时VSMC增殖受到明显抑制,均呈现剂量依赖性。结论：逆转录病毒介导iNOS基因可高效转染体外培养的VSMC,并在细胞内表达活性的iNOS蛋白,而且产生大量的NO,明显抑制VSMC增殖。为iNOS转基因治疗血管移植术后再狭窄的临床应用提供有力的实验依据。     

弱激光对脂质体介导的血管平滑肌细胞基因转染的影响

本研究采用阳离子脂质体介导外源基因转染体外培养的兔血管平滑肌细胞（ＳＭＣ）,在基因转染过程中给予激光照射,用细胞化学染色方法测定基因转染阳性率。结果显示：用５１０．６ｎｍ激光于基因转染前,以功率密度１ｍｗ／ｃｍ２,能量密度２、４、６Ｊ／ｃｍ２和５ｍＷ／ｃｍ２,４、６Ｊ／ｃｍ２;及１０ｍＷ／ｃｍ２,２Ｊ／ｃｍ２进行照射均能显著提高基因转染率（ｐ＜０．０５）;于基因转染后即刻以功率密度１ｍＷ／ｃｍ２、能量密度２Ｊ／ｃｍ２和５ｍＷ／ｃｍ２、６Ｊ／ｃｍ２照射也能提高基因转染率（ｐ＜０．０５）。而用６２７．８ｎｍ激光照射对基因转染率无显著影响     

诱导血红素加氧酶-1表达在器官移植中的保护作用

器官移植术中及术后移植器官的缺血再灌注损伤（ischemia-repeffusion injury,IRI）和免疫排斥反应一直困扰着外科医生.血红素加氧酶-1（heme oxygenase-1,HO-1）是血红素代谢过程中的限速酶,广泛分布于哺乳动物的各种组织细胞中.血红素在它的催化下降解代谢为一氧化碳（CO）、胆绿素和游离铁离子.HO-1在氧化应激、炎性反应、低氧和缺血等状态下均能高度表达.HO-1及其催化血红素代谢产物主要通过抗炎性反应、抗氧化反应、调节同种异体反应性T细胞的活性及增殖、抗内皮细胞凋亡、抑制内皮细胞活化等作用机制,对移植器官起到抗IRI和抗免疫排斥作用,从而增加移植器官成活率及延长其存活时间.     

血红素加氧酶-1重组载体的构建及在胃癌细胞中的初步研究

目的构建人血红素加氧酶-1（hemeoxygenase-1,HO-1）及其突变体的真核表达载体,观察其在胃腺癌细胞中HO-1表达和活性变化,研究HO-1活性变化的胃腺癌细胞对顺铂抗药能力的变化,为进一步研究HO-1对肿瘤细胞影响机制奠定基础。方法根据GenBank中HO-1cDNA序列设计引物,调取基因并克隆入pcDNA3．1（＋）质粒中,构建表达人野生型HO-1与突变型HO-1（HO-1G143H）的重组质粒。脂质体介导重组质粒转染胃腺癌细胞BGC823,用RT—PCR和Western印迹法分别检测细胞中HO-1mRNA的表达和蛋白表达水平,体外测定HO—1活性变化,应用顺铂进行体外抗药性实验。结果酶切鉴定和测序证实,HO-1真核表达载体构建成功;转染质粒后的BGC823,HO-1的mRNA和蛋白的表达水平明显上升;转入野生型质粒的细胞HO-1活性上升,转入突变型质粒的细胞HO-1活性下降;HO-1活性下降BGC823细胞抗顺铂杀伤能力增强。结论构建了HO-1野生型与突变型真核表达载体;将其转入胃腺癌细胞,引起了HO-1的mRNA和蛋白表达的增加和活性变化;体外实验表明,HO-1活性下降的BGC823细胞抗顺铂能力增强。     

血红素加氧酶-1对肝癌细胞细胞周期调控因子的影响

目的观察血红素加氧酶-1（heme oxygenase 1,HO-1）对人肝癌细胞HepG2细胞周期调控因子的影响。方法构建含有野生型和突变型HO-1基因的重组载体pcDNA3．1（＋）-wtHO-1和pcDNA3．1（＋）-mHO-1G143H。利用脂质体介导的方法将构建好的重组载体转染肝癌细胞系HepG2,以空载体转染作为对照组。通过G418筛选建立稳定表达野生型和突变型HO-1的HepG2肝癌细胞系。经半定量RT—PCR、Western印迹检测转染细胞系中HO-1 mRNA和蛋白的表达水平。在HO-1表达改变的稳转细胞系中,利用Western印迹检测转染细胞系中P21、P27蛋白表达水平。结果成功实现了野生型和突变型HO-1在HepG2细胞中的过表达;野生型和突变型HO-1过表达均能诱导抑癌基因p21和p27的表达。结论HO．1过表达诱导抑癌基因p21和p27的表达与血红素分解产物无关。HO-1可能通过其它机制调节p21和p27的表达。     

血红素加氧酶-1、树突状细胞和调节性T细胞在慢性气道炎症中的作用及相互关系

慢性气道炎症是多种肺部疾病的共同病理生理过程,是由多种炎症细胞、炎症介质及细胞因子相互作用所致的气道病变。血红素加氧酶(HO)-1、树突状细胞(DC)和调节性T细胞(Treg)参与了气道炎症并发挥不同的作用,表现在HO-1具有抗炎抗氧化及保护细胞的作用;DC除可导致或持续气道炎症反应外,也具有负向调控作用,可诱导免疫耐受而抑制炎症的发展;而Treg可发挥免疫调抑功能,以此维持免疫稳态及抑制气道炎症。HO-1、DC和Treg相互作用,影响着气道炎症的发生发展。现对三者在气道炎症中的作用及相互关系进行综述。     

脂多糖诱导大鼠主动脉血红素氧合酶-1表达及其对血管反应性的影响

目的：探讨血红素－HO－1－CO－cGMP道路对内毒素血症大鼠主动脉血管张力的影响及其分子机制。方法：用离体血管环张力测定技术,观察静脉注射脂多糖（LPS）6h,大鼠胸主动脉环（TARs)对苯肾上腺素（PE）累积收缩反应。分别用一氧化碳（CO）供体正缺血红素（He),血红素氧合酶－1（HO－1）抑制剂锌原卟啉（ZnPP-IX),鸟苷酸环化酶（sGC)抑制剂亚甲兰（MB）预卵育后,测定TARs对PE收缩反应的变化。分别测定主动脉中CO含量,HO－1活性,Western blot测定HO－1蛋白含量,RT－PCR检测HO－1 mRNA表达的改变。结果：LPS组TARs对PE累积收缩反应明显降低,ZnPP-IX可部分逆转低收缩反应,MB可完全逆转低收缩反应,而用He可加重低收缩反应状态;LPS组动脉组织中CO的含量上升,HO－1活性、蛋白表达量和mRNA表达均明显增加。结论：LPS可使主动脉HO－1基因表达上调,蛋白含量及酶活性明显增加,表明启动血红素－HO－1－CO－cGMP通路,是介导ES大鼠主动脉低收缩反应重要机制之一。     

Overexpression of heme oxygenase-1 protects smooth muscle cells against oxidative injury and inhibits cell proliferation

To investigate whether the expression of exogenous heme oxygenase-1 (HO-1) gene within vascular smooth muscle cells (VSMC) could protect the cells from free radical attack and inhibit cell proliferation, we established an in vitro transfection of human HO-1 gene into rat VSMC mediated by a retroviral vector. The results showed that the profound expression of HO-1 protein as well as HO activity was 1.8- and 2.0-fold increased respectively in the transfected cells compared to the non-transfected ones. The treatment of VSMC with different concentrations of H2O2 led to the remarkable cell damage as indicated by survival rate and LDH leakage. However, the resistance of the HO-1 transfected VSMC against H2O2 was significantly raised. This protective effect was dramatically diminished when the transfected VSMC were pretreated with ZnPP-IX, a specific inhibitor of HO, for 24 h. In addition, we found that the growth potential of the transfected cells was significantly inhibited directly by increased activity of H     

Induction of heme oxygenase-1 is involved in anti-proliferative effects of paclitaxel on rat vascular smooth muscle cells

In this study, we evaluated the possibility that the anti-proliferative effects of paclitaxel on vascular smooth muscle cells (VSMCs) of the rat might be due to the induction of HO-1 gene expression. Treatment of the cells with paclitaxel resulted in marked time- and dose-dependent inductions of HO-1 mRNA, followed by corresponding increases in HO-1 protein expression and HO enzymatic activities. Furthermore, paclitaxel rapidly activated the JNK, ERK, and p38 mitogen-activated protein kinase pathways. A specific inhibitor of JNK, SP600125, abolished paclitaxel-induced HO-1 mRNA expression, whereas PD98059, a specific inhibitor of ERK, and SB203580, a specific inhibitor of p38, had no significant effect. Finally, the suppression of platelet-derived growth factor induced VSMC proliferation was abolished by the HO inhibitor, ZnPP, as well as by the CO scavenger, hemoglobin. These results demonstrated that paclitaxel induces the expression of HO-1 via the JNK pathway in VSMC and that HO-1 expression might be responsible for the anti-proliferative effect of paclitaxel on VSMC.     

Impact of the tissue factor pathway inhibitor gene on apoptosis in human vascular smooth muscle cells

Tissue factor pathway inhibitor (TFPI) plays a vitally important role in the blood coagulation pathway. Recent studies indicated that TFPI induces apoptosis in vascular smooth-muscle cells (VSMCs) in animals. The present study investigated whether the TFPI gene could also induce apoptosis in human vascular smooth-muscle cells (hVSMCs). Such cells were isolated from human umbilical arteries and subsequently transfected with pIRES-TFPI plasmid (2 μg/mL). MTT assaying and cell counting were applied to measure cell viability and proliferation, RT-PCR was utilized to analyze TFPI gene expression in the cells. Apoptosis was analyzed by fluorescence activated cell sorting (FACS). Several key proteins involved in apoptosis were examined through Western blotting. It was shown that TFPI gene transfer led to its increased cellular expression, with a subsequent reduction in hVSMC proliferation. Further investigation demonstrated that TFPI gene expression resulted in lesser amounts of procaspase-3, procaspase-8 and procascase-9, and an increased release of mitochondrial cytochrome c (cyt-c) into cytoplasm, thereby implying the involvement of both extrinsic and intrinsic pathways in TFPI gene-induced apoptosis in hVSMCs.     

Resident phenotypically modulated vascular smooth muscle cells in healthy human arteries

Vascular interstitial cells (VICs) are non‐contractile cells with filopodia previously described in healthy blood vessels of rodents and their function remains unknown. The objective of this study was to identify VICs in human arteries and to ascertain their role. VICs were identified in the wall of human gastro‐omental arteries using transmission electron microscopy. Isolated VICs showed ability to form new and elongate existing filopodia and actively change body shape. Most importantly sprouting VICs were also observed in cell dispersal. RT‐PCR performed on separately collected contractile vascular smooth muscle cells (VSMCs) and VICs showed that both cell types expressed the gene for smooth muscle myosin heavy chain (SM‐MHC). Immunofluorescent labelling showed that both VSMCs and VICs had similar fluorescence for SM‐MHC and αSM‐actin, VICs, however, had significantly lower fluorescence for smoothelin, myosin light chain kinase, h‐calponin and SM22α. It was also found that VICs do not have cytoskeleton as rigid as in contractile VSMCs. VICs express number of VSMC‐specific proteins and display features of phenotypically modulated VSMCs with increased migratory abilities. VICs, therefore represent resident phenotypically modulated VSMCs that are present in human arteries under normal physiological conditions.     

Cell sex determines anoikis resistance in vascular smooth muscle cells

Sexual dimorphism, detectable in vascular smooth muscle cells freshly isolated from aorta of male and female rats, is associated with a different susceptibility to radiation-induced apoptosis. In this work we investigated the mechanism underlying this difference and discovered that, in comparison with cells from male rats, cells from female rats show adhesion-associated resistance to apoptosis, the so called anoikis resistance. This is apparently due to a more adhering phenotype, characterized by a well organized actin microfilament cytoskeleton and to an increased phosphorylated focal adhesion kinase, and, more importantly, to a higher propensity to undergo survival by autophagy.     

Isolation and culture of vascular smooth muscle cells from rat placenta

We developed a new separation method for isolating placental vascular smooth muscle cells (PVSMCs) from a rat in this study. Our method used the magnetic force between a magnet and ferrous ferric oxide (Fe₃O ₄) to make the separation and extraction processes easier and more efficient. From the first to sixth generation, the cells isolated using this protocol were identified as smooth muscle cells (SMCs) by their immunoreactivity to the SMC markers and by the “hill and valley” morphology. PVSMCs were exposed to angiotensin II (1 μmol/L) and resulted in sharply increased intracellular Ca ²⁺ concentration. Furthermore, activation of protein kinase C (PKC) increased concomitantly with a decrease in calponin expression. These results indicate that the isolated cells had biological activity. Our method of isolating PVSMCs from rat leads to isolation of cultured cells with activity and high purity. The approach will be useful in research studies on placental vascular diseases.     

Use of human vessels and human vascular smooth muscle cells in pharmacology

Relatively limited information is available regarding the mechanisms controlling vasomotricity in human vessels. Isolated vessels obtained from patients undergoing surgery were used to characterize the role of endothelial factors and to study coupling mechanisms between receptors, intracellular calcium, and contraction. However, these investigations are limited by the availability of tissues and many uncontrolled factors. Cultured human vascular cells were also used, were these cells rapidly lose at least some of their differentiated characters. Recently, a human blood vessel equivalent was constructed in vitro from cultured cells, using tissue engineering. This technique allowed us to obtain vessel equivalents containing intima, media, and adventitia layers or tubular media layer only. Contraction and rises in intracellular calcium produced by agonists were studied, indicating that such human vessel equivalents may provide valuable models for pharmacological studies.