番茄红素对血管内皮细胞氧化应激损伤的作用及机制研究

目的:观察番茄红素(lycopene,LYC)对于血管内皮细胞功能的作用,探讨其作用机制。方法:人脐静脉内皮细胞(HUVECs)处理实验分组:对照组,H2O2组,H2O2+LYC组(1、2、4、8μmolL-1)。MTT法检测HUVECs存活率;免疫印迹法(Western blot)检测p38MAPK蛋白磷酸化水平、抗凋亡蛋白B淋巴细胞/白血病-2(bcl-2)及线粒体凋亡通路相关蛋白bax的表达;细胞黏附能力测定和伤口愈合实验检测HUVECs粘附率和迁移率;TUNEL法检测HUVECs凋亡率;ELASA法测定HUVECs内活性氧(ROS),超氧化物歧化酶(SOD),乳酸盐脱氢酶(LDH)释放量和caspase-3的活性。结果:H2O2损伤后HUVECs存活率显著降低(P0.01),凋亡率显著增加(P0.01),黏附和迁移能力显著降低(P0.01),bax和p-p38MAPK的表达上调,bcl-2的表达下调,并且ROS、LDH的释放和caspase-3的活性增加(P0.01),SOD的释放减少。而LYC的预处理可以明显逆转H2O2以上作用。结论:H2O2氧化应激损伤中,LYC保护内皮细胞可能与其抗过氧化损伤细胞凋亡,抑制异常的p38MAPK信号通路有关。     

miR-29b对TNF—α诱导的人脐静脉内皮细胞增殖与凋亡的影响

目的探索过表达miR-29b对TNF-α 诱导的人脐静脉内皮细胞（HUVECs）增殖与凋亡的影响及初步作用机制。方法MTT 法筛选TNF—α诱导HUVECs的最佳浓度和时间,建立细胞凋亡模型;MTY法筛选miR-29bmimic转染HUVECs的最佳转染时间和浓度：MTr法检测过表达miR-29b对TNF-α诱导HUVECs增殖活力的影响;Hoechst33342荧光染色检测过表达miR-29b对TNF—α诱导HUVECs凋亡的影响：Western印迹技术检测过表达miR-29b对Akt磷酸化水平、Bcl-2蛋白表达的影响。结果TNF—α诱导的HUVECs凋亡的最佳浓度为10ng／ml,最佳时间是48h;miR-29bmimics转染HUVECs的最佳浓度为50nmol/L,最佳作用时间是48h;过表达miR-29b能显著降低TNF-α诱导的HUVECs的增殖活力（P〈0．001）;Heochst33342荧光染色结果显示。miR-29b过表达能促进TNF-α诱导的HUVECs的凋亡（P〈0．05）;过表达miR-29b能显著下调Akt磷酸化（p-Akt）与Bcl-2蛋白的表达（P〈0．001）。结论过表达miR-29b可降低TNF-α诱导的HUVECs的增殖活力并促进其凋亡,其机制可能与下调Akt磷酸化、Bd-2蛋白的表达相关。     

三氧化二砷对人脐静脉内皮细胞凋亡及VCAM-1/ICAM-1 表达的影响

目的:观察三氧化二砷(As2O3)对血管内皮细胞增殖、凋亡及VCAM-1/ICAM-1表达的影响,探讨As2O3对血管内皮细胞增殖生长以及炎症反应的影响。方法:人脐静脉内皮细胞(HUVEC)体外培养,以不同As2O3浓度及时间对其进行干预。采用CCK-8测定细胞增殖活性,流式细胞仪AnnexinⅤ/PI双染法检测细胞的凋亡率,实时荧光定量PCR检测VCAM-1mRNA表达,酶联免疫吸附试验(ELISA)检测细胞间黏附分子(VCAM-1)及血管细胞黏附分子(ICAM-1)的表达情况。结果:当As2O3浓度在3μmol.L-1时HUVEC培养24 h的的凋亡率为(0.134±0.03)%,48 h为(3.305±0.53)%,72 h为(3.748±0.84)%(P<0.05),凋亡率均在一较低水平。当As2O3浓度>3μmol.L-1时HUVEC凋亡率明显增加(P<0.01)。不同浓度As2O3作用HUVEC48 h后检测上清液中ICAM-1与VCAM-1浓度时发现1μmol.L-1时VCAM-1表达即开始增加(123.32±3.78 mmol.L-1,P<0.01),而HUVEC表达ICAM-1含量与对照组相比差异并不明显(38.94±2.59 mmol.L-1,P>0.05),随着As2O3浓度的增加,HUVEC表达ICAM-1/VCAM-1的量均增加但敏感性不同。对照组及(1.0、2.0、3.0、4.0、5.0)μmol.L-1As2O3作用于HUVEC 48 h实时荧光定量PCR法检测VCAM-1mRNA表达量明显增加,与对照组相比实验组的表达量分别为(1.657±0.287,1.858±0.241,2.321±0.280,3.012±0.235,3.508±0.342)(P<0.01)。结论:As2O3可直接降低细胞活性,诱导细胞凋亡,并且呈一定的时间-浓度依赖性。在较低浓度时VCAM-1/ICAM-1的表达在一个相对较低的水平,随着As2O3浓度的逐渐升高,内皮细胞凋亡率增高,VCAM-1/ICAM-1表达增加,并且VCAM-1/ICAM-1对As2O3的敏感性呈现一定的差异性。     

红杉醇对高糖诱导的人脐静脉内皮细胞NOX4、eNOS表达的影响

目的：观察红杉醇（Scq）对高糖诱导的人脐静脉内皮细胞（HUVECs）损伤的保护作用及机制。方法：原代培养HUVECs,红杉醇（0．1,1,10μmol/L）预处理1h后,30mmol/L葡萄糖诱导内皮细胞损伤。5-溴脱氧尿嘧啶核苷（BrdU）掺入法检测细胞增殖,流式细胞术检测细胞周期,2’7’-二乙酰二氯荧光素（DCFH-DA）免疫荧光法检测细胞内活性氧簇（R0s）水平,比色法检测细胞-氧化氮（NO）、丙二醛（MDA）及过氧化氢（H202）水平,real-timePCR和Westernblot检测细胞内皮型一氧化氮合酶（eNos）及NADPH氧化酶4（NOX4）mRNA和蛋白表达。结果：Seq预处理1h后能明显减轻高糖诱导的血管内皮细胞损伤,促进细胞增殖,降低胞内NOX4的表达及ROS、MDA及H202水平,上调eNOS的表达及NO水平。结论：Seq对高糖诱导的内皮细胞损伤具有一定的保护作用,其机制可能与其抗氧化、上调eNOS的表达有关。     

TWIST1在人脐静脉内皮细胞增殖、迁移及血管形成中的作用

探讨TWIST1在原代人脐静脉内皮细胞（human umbilical vein endothelial cells,HUVECs）增殖、迁移及体外血管生成中的作用。用有靶向人TWIST1基因shRNA（pLL3.7-shTwist1-GFP）的慢病毒液感染试验组细胞,同时以携带Scramble shRNA的慢病毒液（pLL3.7-shCtrl-GFP）感染对照组细胞,用流式细胞术测定细胞感染效率,实时荧光定量PCR（real-time fluorescent quantitative PCR,qRT-PCR）检测shRNA的基因沉默效率。通过制作细胞生长曲线、Annexin V/7AAD染色流式细胞术、细胞划痕实验、小管形成实验、qRT-PCR检测TWIST1表达降低对HUVECs的增殖、凋亡、迁移、血管形成能力以及血管生长因子受体2（vascular endothelial growth factor receptor 2,VEGFR2）基因表达的影响。试验组TWIST1基因表达下降为对照组的30%,表明shTWIST1能有效降低TWIST1基因的表达。与对照组相比,敲降TWIST1能明显抑制HUVECs的增殖（P<0.01）,诱导细胞凋亡（P<0.05）。试验组HUVECs划痕愈合率、体外生成的血管样结构数目和总小管分支长度均显著低于对照组（P<0.01）;与对照组相比,试验组HUVECs中VEGFR2的表达显著降低（P<0.01）。通过探究HUVECs表达的TWIST1在内皮细胞增殖、存活、迁移和毛细血管样结构的形成中的作用,为TWIST1作为抑制肿瘤血管新生治疗的新靶点提供一定的理论依据。     

解脲脲原体诱导脐静脉血管内皮细胞凋亡的研究

目的通过探讨解脲脲原体(Ureaplasma urealyticum,UU)诱导人脐静脉内皮细胞(human umbilicalvein endothelial cells;HUVEC)凋亡的情况,揭示妊娠期间UU垂直传播影响胚胎发育的可能机制。方法不同剂量血清4型UU标准菌株刺激体外培养的HUVEC,通过Annexin-V.FITC/PI双染流式细胞术和DNA Ladder实验观察细胞凋亡情况。结果对照组细胞凋亡率小于各实验组(P<0.01~0.05),其凋亡率和刺激剂量、时间之间呈现一定的剂量-时间-效应关系(P>0.05)。结论UU可诱导人脐静脉内皮细胞凋亡,提示妊娠期间UU感染可能通过诱导脐静脉内皮细胞凋亡破坏胎盘屏障而影响胚胎发育。     

CCR2介导MCP-1诱导的人内皮细胞凋亡

本室前期工作发现,单核细胞趋化蛋白-1(MCP-1)可诱导人内皮细胞(hVECs)凋亡.为进一步揭示MCP-1诱导凋亡分子机理,首先观察MCP-1对hVECs CC类趋化因子受体2(C-C motifchemokine receptor-2,CCR2)蛋白表达的影响.Western印迹结果显示,MCP-1以剂量依赖方式诱导CCR2在hVECs的表达.以脂质体为载体的CCR2反义寡核苷酸序列转染hVECs后,激光共聚焦显微镜及膜联蛋白(annexin)V-FITC/PI双染流式细胞术显示,CCR2反义寡核苷酸转染hVECs48h后可明显降低CCR2蛋白质的表达(P0.05),抑制MCP-1诱导的hVECs凋亡(P0.01).反义CCR2抑制凋亡结果与加入CCR2阻断剂RS102895后细胞凋亡测定结果一致.上述结果表明,MCP-1的主要受体CCR2介导了MCP-1诱导的hVECs凋亡.     

CTSB 对人脐静脉血管内皮细胞系增殖及凋亡的影响

目的:探索组织蛋白酶B(CTSB)对人脐静脉血管内皮细胞系(HUVEC)增殖及凋亡的影响。方法:采用细胞转染法得到慢病毒空载体转染HU-MOCK细胞及CTSB过表达的HU-CTSB细胞,与正常的HUVEC细胞进行比较。采用MTT法检测HUVEC增殖情况;流式细胞仪检测各组细胞凋亡情况;采用蛋白免疫印迹法检测Bal-2/Bax及Caspase家族(Caspase-3/9)的表达情况。结果:HU-CTSB组各时间点HUVEC的增殖率明显低于其他两组,且差异具有统计学意义(P0.05);HU-CTSB组的HUVEC早期凋亡率(右下象限)和晚期凋亡率(右上象限)显著高于HUVEC组和HU-MOCK组,且组间差异具有统计学意义(P0.05);HU-CTSB组的抑制凋亡蛋白Bcl-2表达量明显低于其他两组,且促凋亡蛋白Bax表达量显著高于其他两组,差异均具有统计学意义(P0.05);HU-CTSB组caspases-3和caspases-9的活化片段显著高于其他两组,差异均具有统计学意义(P0.05)。结论:CSTB通过激活Caspase-3/9信号通路来上调Bax,并下调Bal-2,显著抑制HUVEC的增殖,并促进其凋亡,进而影响心血管疾病的发展。。     

脂联素在高糖孵育血管内皮细胞中的表达调节

目的:探讨脂联素(APN)在高糖孵育血管内皮细胞中的表达调节及意义.方法:用不同浓度葡萄糖(5.5、11、22及33mM)孵育人脐静脉内皮细胞(HUVECs),2天后收集细胞和上清液.用实时荧光定量RT-PCR方法检测细胞中APN、肿瘤坏死因子α(TNFα)和过氧化物酶体增生物激活受体γ(PPARγ)的mRNA表达,用ELISA方法检测细胞上清液中APN和TNFα分泌情况.结果:随培养液中葡萄糖浓度的升高,HUVECs中APN的mRNA表达及上清中APN蛋白含量降低(p<0.05),TNFα的mRNA表达及上清中TNFα蛋白含量增加(p<0.05),HUVECs中PPARγ的mRNA表达降低(p<0.05),且组间比较差异均有统计学意义(p<0.05).结论:高糖诱导血管内皮细胞保护性细胞因子APN表达分泌下降,可能是高糖诱导的血管内皮细胞功能损害的重要机制之一.     

原子力显微镜研究高乌甲素对人脐静脉内皮细胞形态的影响

在纳米量级上探测药物对人脐静脉内皮细胞(HUVEC)的抑制作用对于揭示药物的功效及肿瘤的有效治疗十分重要.通过高分辨率的原子力显微镜研究了不同浓度的高乌甲素培养的HUVEC的形貌特征,包括整个细胞的形貌和超微结构的表面膜的差异.从形貌学方面探讨了高乌甲素对HUVEC的抑制作用,可为临床应用高乌甲素提供依据.结果表明,我...     

秦皮甲素对AGEs致损的ECV304细胞增殖及氧化应激的影响

为研究秦皮甲素对血管内皮细胞的保护作用,采用CCK-8法观察秦皮甲素对体外AGEs培养的人脐静脉内皮细胞增殖的影响。检测不同浓度AGEs以及秦皮甲素作用后对内皮细胞一氧化氮(NO)、不对称二甲基精氨酸(ADMA)水平的影响以及内皮细胞氧化应激有关指标:活性氧簇(reactive oxygen species,ROS)、丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD);脂肪代谢相关指标:乳酸脱氢酶(lactic dehydrogenase,LDH)、总胆固醇(total cholesterol,CHO)、甘油三酯(triglyceride,TG)和低密度脂蛋白(low density lipoprotein,LDL),同时分别检测粘附相关因子:血管细胞粘附分子-1(VCAM-1)和细胞间粘附分子-1(ICAM-1)的表达水平。结果显示200 mg/L AGEs对人内皮细胞ECV304增殖有显著抑制作用,秦皮甲素可对抗AGEs导致的内皮细胞增殖抑制,并呈浓度依赖性。在25 mg/L时,保护效应达到最高。秦皮甲素可抵抗ROS生成。同时可改善细胞的脂类代谢:胆固醇、LDL以及TG含量在秦皮甲素作用后改善明显。秦皮甲素可显著抑制内皮粘附因子VCAM-1的表达。秦皮甲素还可上调NO水平,下调ADMA水平。总之,秦皮甲素可有效促进人血管内皮细胞增殖并在改善氧化应激,脂代谢,粘附因子和NO释放等方面发挥作用。     

脂联素抑制t-BHP诱导的内皮细胞凋亡作用及其可能机制探讨

目的：探讨重组脂联素对第三丁基过氧化氢（t-BHP）诱导的人脐静脉内皮细胞（HUVECs）凋亡的影响及其相关的分子机制。方法：以HUVECs作为研究对象,给予t-BHP处理,模拟体外HUVECs氧化损伤细胞凋亡模型。在此基础上,用携带重组脂联素基因的腺病毒转染HUVECs,观察重组脂联素对t-BHP诱导的HUVECs凋亡的影响。用MTT法检测细胞增殖活力。Hochest/PI荧光染色检测细胞凋亡率。Western blot检测细胞凋亡相关蛋白p-JNK、JNK和Caspase 3表达水平的变化。结果：100 μmol/L的t-BHP作用8 h可诱导HUVECs发生凋亡。与对照组相比,t-BHP组p-JNK、active caspase 3表达增多（P<0.01）。HUVECs高表达重组脂联素基因后,可明显抑制t-BHP诱导的HUVECs凋亡（P<0.01）,下调t-BHP诱导的p-JNK、active caspase 3表达。结论：持续t-BHP氧化损伤可诱导HUVECs发生凋亡。重组脂联素可有效抑制t-BHP诱导的HUVECs凋亡,其机制与p-JNK、active caspase 3的表达下调有关。     

Paeoniflorin attenuates oxidized low-density lipoprotein-induced apoptosis and adhesion molecule expression by autophagy enhancement in human umbilical vein endothelial cells

Oxidized low-density lipoprotein (ox-LDL)-induced endothelial dysfunction is recognized as a driving force in the development of atherosclerosis (AS). Paeoniflorin (Pae), a typical traditional herbal medicine, possesses anti-inflammatory, antioxidative, antihyperglycaemic, and antiapoptotic properties. Our study aimed to investigate the effects of Pae on ox-LDL-induced injury of the human umbilical vein endothelial cells (HUVECs) and to explore its molecular mechanism. We found that ox-LDL stimulation inhibited cell viability, activated autophagy, and induced apoptosis and adhesion molecule expression in HUVECs. Pae rescued ox-LDL-induced viability reduction and enhanced the ox-LDL-induced autophagy activation in HUVECs. Pae inhibited ox-LDL-induced apoptosis and adhesion molecule expression by autophagy enhancement in HUVECs. In addition, inhibition of SIRT1 by EX-527 abolished the promoting effect of Pae on autophagy and restored the inhibitory effect of Pae on apoptosis and adhesion molecule expression in the presence of ox-LDL. In conclusion, Pae attenuated ox-LDL-induced apoptosis and adhesion molecule expression by autophagy enhancement via upregulation of SIRT1 in HUVECs, shedding light on the mechanism underlying the protective effect of Pae on ox-LDL-induced injury of HUVECs.     

Different expression of NOS isoforms in early endothelial progenitor cells derived from peripheral and cord blood

Cord blood and peripheral-adult blood were compared as different sources of early endothelial precursor cells (eEPCs). Total mononuclear cells (MNCs) were obtained from both blood types and committed to eEPCs by exposure to fibronectin, VEGF, IGF-I, and bFGF. Under this condition, MNCs seeded at the density of 3 x 10(5) cells/cm(2) assumed a spindle shape, which was indicative of developing eEPCs, and expanded in a similar manner irrespective to the blood sources. Ulex europaeus agglutinin (UEA-1) and acetylated low density lipoprotein (acLDL) double staining was present in 90% in both peripheral- and cord-blood eEPCs after 2-week expansion. Also, the ability of eEPCs to form tubule-like structures in Matrigel was independent of their blood source, but dependent on the presence of human umbilical vein endothelial cells (HUVECs). eNOS and nNOS were not detectable by Western blotting in both peripheral and cord-blood eEPCs upon 3 weeks and their mRNA levels were lower than 2% relative to those present in HUVECs. On the contrary, iNOS protein was detectable in peripheral-blood eEPCs, but not in cord-blood eEPCs and HUVECs, as well as iNOS mRNA was more concentrated in peripheral-blood eEPCs than in cord-blood eEPCs and HUVECs. These data suggest that: (a) peripheral and cord blood can be considered comparable sources of eEPCs when they are expanded and differentiated in a short-term period; (b) the extremely low expression of constitutive NOS isoforms in the eEPCs of both blood types should markedly reduce their ability to regulate NO-dependent vasorelaxation; (c) the presence of iNOS in peripheral-blood eEPCs could improve the process of vasculogenesis.     

Retracted: Propranolol suppresses HUVEC viability,migration, VEGF expression,and promotes apoptosis by downregulation of miR-4295

Infantile hemangioma (IH) is a common benign tumor. Human umbilical vein endothelial cells (HUVECs) have the potential of stem cells, which has been widely used in vascular endothelial cell experiments. Oral propranolol was first reported to treat hemangioma in 2008. However, the role of propranolol in IH remains unclear. Therefore, in this study, we investigated the effects of propranolol on HUVECs in vitro, to explore the underlying mechanism of propranolol in IH. HUVECs were treated with 0.15, 1.5, and 15 μM of propranolol, and transfected with microRNA-4295 (miR-4295) mimic. Cell viability, migration, and apoptosis were examined using Cell Counting Kit-8, transwell assay, and flow cytometry analysis, respectively. In addition, the expressions and concentrations of miR-4295, vascular endothelial growth factor (VEGF), VEGF-A, FLT1, FLT2, and FOXF1 were assessed using real-time polymerase chain reaction, Western blot assay, and enzyme-linked immunosorbent assay. We found that 15 μM of propranolol decreased HUVEC viability the most. Then, cell migration and the concentrations of VEGF and VEGF-A were reduced, and apoptosis was increased when treated with propranolol. Meanwhile, the expressions of VEGF, VEGF-A, FLT1, FLT2, and FOXF1 were downregulated by propranolol exposure. Further study showed that miR-4295 expression was upregulated in IH tissues, and propranolol treatment downregulated miR-4295 expression in HUVECs. MiR-4295 overexpression alleviated the reductions of viability, migration, and factors expression, as well as the increase of apoptosis. Propranolol suppressed HUVEC viability, migration, the expression of VEGF, VEGF-A, FLT1/2, FOXF1, and promoted apoptosis via downregulation of miR-4295. This study lays a foundation for further study of the effect of propranolol on IH.     

Hypoxia-induced apoptosis in endothelial cells and embryonic stem cells

Background: To evaluate the influence of hypoxia and molecular events in endothelial and embryonic stem cells.Materials and Methods: Human umbilical vein endothelial cells (HUVECs) and mouse embryoid body (EB) cells were subjected to hypoxic conditions for different time courses. DNA fragmentation assay, quantification of apoptotic cells by TUNEL assay measured by flowcytometry, and Western blot analysis for the molecular events of apoptosis were performed.Results: DNA fragmentation could be identified under hypoxic conditions in HUVECs and mouse EBs. The DNA fragmentation increased when the hypoxic interval was extended.In situ internucleosomal DNA fragmentation-TUNEL assay also found that the percentages of apoptotic cells increased gradually in HUVECs and mouse EBs when the hypoxic interval was extended. Furthermore, the levels of expression of p53 and Bax both increased in hypoxic conditions.Conclusions: Hypoxia increases both HUVEC and mouse EB apoptosis, which is associated with increase in p53/Bax expression.     

信号转导和转录活化因子3通过趋化因子CX3C配体1促进血管内皮细胞增殖迁移

信号转导和转录活化因子3 (STAT3)与趋化因子CX3C配体1 (Fractalkine/CX3CL1)在血管炎症和损伤中起重要作用,为了探讨STAT3是否通过CX3CL1促进血管内皮细胞增殖和迁移,在血管内皮细胞(HUVEC)中过表达或敲降STAT3,通过quantitative real-time PCR、Western blotting实验确定STAT3对CX3CL1表达的影响。构建含有STAT3结合位点及突变STAT3结合位点的CX3CL1启动子荧光素酶报告基因质粒,利用荧光素酶活性分析实验研究STAT3对CX3CL1启动子转录活性的作用。利用MTT实验检测过表达或敲降STAT3或CX3CL1对血管内皮细胞增殖率的影响。利用划痕实验检测过表达或敲降STAT3或CX3CL1对血管内皮细胞迁移率的影响。结果显示,过表达STAT3可以促进CX3CL1表达,敲降STAT3可以使CX3CL1表达下调。STAT3可以直接结合到CX3CL1的启动子促进其转录激活,其促进作用依赖于CX3CL1启动子上的GAS位点。敲降STAT3可以抑制血管内皮细胞的迁移,过表达CX3CL1拮抗该抑制作用。总结得出,STAT3通过结合到CXCL1启动子促进CX3CL1转录与表达进而促进血管内皮的增殖与迁移。