三角波形低频脉冲磁场对大鼠心肌微血管内皮细胞增殖、迁移和细胞周期的影响

目的: 研究在固定时间和频率下,三角波形的低频脉冲磁场(LF-PMF)对大鼠心肌微血管内皮细胞（CMECs）增殖、迁移以及细胞周期的影响。方法: 实验分为4组（对照组,1.0mT组,1.4mT组,1.8mT组）。对照组不加磁场干预;其余各组分别在频率为15 Hz,磁场强度分别为1.0mT、1.4mT和1.8mT,时间为4 h/d,连续照射3 d的条件下,用三角波形作用离体培养的大鼠CMECs。利用MTT法检测细胞增殖情况,transwell法观察细胞迁移状况,流式细胞仪技术检测细胞周期变化。结果: 1.4mT和1.8mT组磁场促进CMECs增殖的作用差异显著［(0.200±0.043)A值, vs.(0.159±0.037)A值,(0.225±0.042)A值,vs.(0.159±0.037)A值,P<0.05］。迁移能力与对照组相比也有显著提高［(27.20±4.76)个/视野 vs.(22.60±4.77)个/视野,(33.80±3.19)个/视野 vs.(22.60±4.77)个/视野,P<0.05］。CMECs在细胞周期中的分布发生改变,DNA合成期(S期)和合成后期(G2期)细胞的比例增加。1.0mT组LF-PMF胞对细胞增殖、迁移以及细胞周期的影响均不明显。结论: 低频脉冲磁场磁场对CMECs的生物学作用与磁场的强度相关,1.4mT和1.8mT组LF-PMF促进细胞增殖、迁移及使其DNA合成的能力提高。     

辛伐他汀对雷帕霉素作用下的大鼠心肌微血管内皮细胞的影响

目的:探讨辛伐他汀(SIM)对雷帕霉素(RAPA)作用下大鼠心肌微血管内皮细胞(CMECs)增殖、迁移、凋亡及一氧化氮(NO)分泌的影响。方法:用不同浓度(0、0.01、0.1、1及10μg/L)的RAPA处理CMECs 24 h,分别采用MTT比色法及transwell法检测细胞的增殖能力和迁移能力,并选择合适干扰浓度(抑制效果居中,即对细胞的增殖及迁移有一定的抑制作用,但不会完全抑制,选择浓度为1μg/L)。对已加入RAPA(1μg/L)的细胞中加入不同浓度(0、0.01、0.1、1及10μmol/L)的SIM,共孵育24 h,采用MTT比色法检测细胞的增殖能力,用transwell法检测细胞迁移能力,用Hoechst染色法计算细胞的凋亡率,用Griess反应检测NO的分泌活性。结果:与对照组相比较,单纯以RAPA干预细胞后,细胞增殖和迁移的能力均显著下降(P0.05,P0.01),且呈浓度依赖性。而在RAPA基础上加入不同浓度的SIM后,与单纯RAPA组比较,细胞的增殖能力及迁移能力均显著增强(P0.05,P0.01),NO的分泌活性明显升高(P0.05),细胞的凋亡率明显下降(P0.05)。结论:RAPA能抑制CMECs增殖及迁移、NO分泌并诱导其凋亡。而将CMECs与SIM共孵育24 h后,对RAPA诱导的细胞损伤具有抑制作用。     

不同剂量阿司匹林对大鼠心肌微血管内皮细胞血管新生功能的影响

目的探讨不同剂量阿司匹林对大鼠心肌微血管内皮细胞(CMECs)血管新生功能的影响。方法 4周龄雄性SPF级SD大鼠,处死后分离右心室,提取原代心肌微血管内皮细胞进行培养,依据阿司匹林浓度不同分为对照组、实验组(1、10、100、1 000、5 000μmol/L),分别检测其对CMECs的增殖能力、细胞迁移能力和纤维连接蛋白(FN)水平的影响。结果培养48 h后,阿司匹林1、10、100μmol/L组与对照组比较细胞量和凋亡指数(AI)值均无统计学意义(P0.05),仅浓度为1 000和5 000μmol/L组与对照组比较细胞量和AI值差异显著(P0.05);划痕实验显示1、10、100μmol/L阿司匹林组的划痕间距均显著小于对照组,10、100μmol/L阿司匹林组的Transwell迁移数显著高于对照组,10、100μmol/L阿司匹林组的成管个数显著高于对照组,5 000μmol/L阿司匹林组的成管个数显著低于对照组(P0.05),其余组的相应指标与对照组无统计学意义(P0.05);FN的分析结果显示除1μmol/L阿司匹林组外其余浓度组均显著高于对照组(P0.05)。结论阿司匹林对CMECs作用低浓度时表现为促进细胞增殖和迁移作用,并能显著诱导FN分泌;高浓度时表现为抑制增殖和迁移作用,但是对FN的分泌具有促进作用。     

17β-雌二醇对大鼠心肌的促血管新生作用:促进微血管内皮细胞分泌VEGF

目的: 研究17β-雌二醇（17β-E2）对大鼠心肌微血管内皮细胞(CMEC)的促血管新生作用。方法: 以体外培养的CMECs为模型，用免疫荧光染色法检测雌激素受体(ER)的表达。用MTT比色法检测不同剂量的17β-E2对细胞增殖的影响。用细胞划痕法检测加入17β-E2后细胞的迁移能力。用Millicell小室测定法检测17β-E2对细胞侵入能力的影响。用管样结构形成实验观察加入17β-E2后细胞分化的情况。用ELISA法检测17β-E2对细胞分泌血管内皮生长因子（VEGF）的影响。结果: 免疫荧光染色法检测显示，心肌微血管内皮细胞内存在ER。MTT比色法检测表明，17β-E2可明显促进CMECs的增殖，在其浓度为0.01 μmol/L时，可产生最大的增殖效应；而雌激素的拮抗剂三苯氧胺（TMF）则可阻断此效应。同时发现，17β-E2处理组CMECs的迁移能力和管样结构形成的能力，均明显强于对照组及17β-E2+TMF处理组。与对照组及17β-E2+TMF处理组相比，17β-E2处理组可明显促进大鼠CMESs分泌VEGF（P<0.01）。结论: 在体外环境中，17β-E2可刺激CMECs自分泌VEGF，提高CMECs血管新生的活性。     

阿司匹林对心肌微血管内皮细胞血管新生功能的影响

目的:探讨不同剂量阿司匹林对大鼠心肌微血管内皮细胞（CMECs）血管新生功能的影响和可能的机制。方法:消化法分离大鼠CMECs,用不同浓度（1、10、100、1 000和5 000 μmol/L）的阿司匹林处理体外培养的大鼠CMECs后,分别用CCK-8比色法检测细胞增殖、TUNEL法检测细胞凋亡、划痕试验及Transwell小室评价细胞迁移能力、成管实验评价血管新生能力、Western blot法检测蛋白激酶B（AKT）磷酸化水平,ELISA法测定纤维连接蛋白（FN）表达。结果: ①1、10和100 μmol/L的阿司匹林对,CMECs的增殖、凋亡和AKT磷酸化水平无明显影响,1000、5000μmol/L的阿司匹林对组显著抑制CMECs增殖、下调AKT磷酸化水平、诱导细胞凋亡;②10、100 μmol/L的阿司匹林促进CMECs迁移、成管;③FN表达随阿司匹林浓度升高而升高。结论:①低浓度阿司匹林（1~100μmol/L）对CMECs的存活无明显影响,而高浓度的阿司匹林（1 000、5 000 μmol/L）显著抑制CMECs增殖、下调AKT磷酸化水平、细胞凋亡增多。②低浓度阿司匹林（1~100 μmol/L）作用下,阿司匹林促进CMECs迁移、成管能力,可能与阿司匹林作为环氧化酶（COX）抑制剂,减少前列环素（PGI2）生成引起FN表达上调有关。     

同型半胱氨酸对大鼠心肌微血管内皮细胞线粒体功能的影响及通心络的保护作用

目的探讨通心络(TXL)对同型半胱氨酸(Hcy)诱导大鼠心肌微血管内皮细胞线粒体功能损伤的保护作用及机制。方法用Hcy建立大鼠心肌微血管内皮细胞损伤模型,分为对照组、Hcy组、TXL-L组、TXL-M组、TXL-H组。分别检测各组细胞线粒体活性、线粒体膜电位(MMP)和细胞内活性氧(ROS)水平。结果与对照组相比,模型组细胞线粒体活性和MMP降低,细胞内ROS水平升高(P<0.05,P<0.01);与模型组相比,TXL各浓度组均能不同程度地改善上述指标变化(P<0.05,P<0.01)。结论 TXL能够改善Hcy诱导的大鼠心肌微血管内皮细胞线粒体功能障碍,其作用机制可能与降低细胞内ROS水平有关。     

促红细胞生成素衍生肽抑制大鼠心肌微血管内皮细胞缺血再灌注损伤的作用研究

目的:探讨促红细胞生成素衍生肽(HBSP)抑制大鼠心肌微血管内皮细胞(CMECs)缺血/再灌注损伤的作用及其机制。方法:分离培养大鼠CMECs,建立缺血/再灌注损伤模型,随机分为对照组、模拟缺血/再灌注组(SI/R组)、SI/R+重组人促红细胞生成素组(SI/R+rh EPO组)、SI/R+HBSP组;HBSP分别选择2.5 ng/ml、25 ng/ml、50 ng/ml、100 ng/ml四个浓度,通过噻唑蓝(MTT)比色实验检测CMECs增殖能力以确定药物作用的最适浓度。而后采用细胞划痕实验检测细胞迁移能力,末端脱氧核苷酸转移酶介导的d UTP缺口末端标记测定(TUNEL)法检测细胞凋亡率来进一步验证其保护作用。研究保护机制的实验分组为:对照组、SI/R组、SI/R+HBSP(HBSP最适浓度为25 ng/ml)组、SI/R+HBSP+磷脂酰肌醇-3激酶(PI3K)抑制剂(LY294002)组、SI/R+HBSP+雷帕霉素组,采用蛋白免疫印迹(Western blot)法检测蛋白激酶B(AKT)、哺乳动物雷帕霉素靶蛋白(m TOR)、核糖体S6蛋白激酶(p70S6K)等蛋白的表达及其磷酸化水平。结果:与SI/R组比,SI/R+rh EPO组和SI/R+HBSP组CMECs增殖能力明显上升(P<0.05),凋亡率显著下降(P<0.01),迁移能力增强。用抑制剂LY294002及雷帕霉素处理后,与SI/R组相比,SI/R+HBSP组AKT、m TOR及p70S6K的磷酸化水平均显著提高(P<0.05);与SI/R+HBSP组相比,SI/R+HBSP+LY294002组的AKT、m TOR及p70S6K的磷酸化水平均显著下降(P<0.05),SI/R+HBSP+雷帕霉素组m TOR磷酸化水平及p70S6K磷酸化水平显著下降(P<0.05)。结论:HBSP能够抑制缺血/再灌注诱导的CMECs的损伤,其保护作用可能与PI3K-AKT/mT OR信号通路激活有关。     

Ghrelin对同型半胱氨酸诱导大鼠心肌微血管内皮细胞损伤和炎性反应的保护作用

目的探讨Ghrelin对同型半胱氨酸(Hcy)诱导大鼠心肌微血管内皮细胞(CMECs)损伤和炎性反应的保护作用及作用机制。方法取雄性SD大鼠心肌细胞培养鉴定后,分别以不同浓度的Hcy、不同浓度Ghrelin孵育24 h,采用MTT法检测细胞活力。另选细胞随机分为对照组、Hcy组(0.25 mmol/L)、Ghrelin组(100 ng/L)和混合组,Hoechst染色计算细胞凋亡率,NO试剂盒检测NO的分泌活性,ELISA检测白细胞介素6(IL-6)、细胞间黏附分子1(ICAM-1)的分泌,免疫印迹法检测NF-κB蛋白的表达。结果不同浓度Hcy细胞活性较不同浓度Ghrelin明显降低(P0.05,P0.01)。与对照组比较,Hcy组NO的分泌活性显著降低,细胞凋亡率显著上升,IL-6和ICAM-1的分泌显著增加,NF-κB的表达显著增加,差异有统计学意义(P0.05,P0.01)。与Hcy组比较,混合组可以明显抑制上述指标(P0.05)。结论 Hcy可诱发大鼠CMECs的损伤和炎性反应,而Ghrelin预处理对Hcy诱导的损伤及炎性反应有明显的抑制作用,其保护作用机制可能与抑制NF-κB信号通路有关。     

芪苈强心提取物对缺氧大鼠心肌微血管内皮细胞VEGF及HIF-1α表达作用的研究

目的观察芪苈强心提取物对缺氧诱导的大鼠心肌微血管内皮细胞VEGF及其上游调控因子HIF-1α表达的影响,探讨芪苈强心胶囊抗心力衰竭作用的相关机制。方法植块法培养2周龄SD大鼠心肌微血管内皮细胞并鉴定,传代后将第二代细胞随机分为三组：空白对照组,缺氧干预组,缺氧干预＋芪苈强心组。干预6h后收集细胞上清,提取细胞核蛋白及胞浆蛋白,利用ELISA,WesternBlot分别检测细胞上清液中VEGF含量及细胞内VEGF上游调控因子HIF-1α的表达。结果与对照组比较,缺氧干预组心肌微血管内皮细胞VEGF、HIF-1α表达增加,与缺氧干预组比较,芪苈强心干预组VEGF含量均进一步升高,HIF-1α也表现出相同趋势。结论芪苈强心提取物能够通过促进缺氧状态下心肌微血管内皮细胞HIF-1α诱导的VEGF表达,这可能是芪苈强心发挥抗心力衰竭作用的机制之一。     

凝血酶致脑微血管内皮细胞凋亡作用

脑微血管内皮细胞(brain microvascular endothelial cell,BMVEC)在脑疾病发生、发展中占重要地位[1].近年来,BMVEC的体外培养方法已获成功[2].凝血酶是一种血清丝氨酸蛋白酶,除参与凝血外,它还促进BMVEC收缩并增加其通透性[3],并与肺动脉内皮细胞及神经元凋亡相关[4～6].本实验用体外原代培养的大鼠BMVEC研究凝血酶的作用,并用其受体拮抗剂水蛭素探讨致病机制.     

Aprotinin impairs coronary endothelial function and down-regulates endothelial NOS in rat coronary microvascular endothelial cells

OBJECTIVE: The non-specific serine protease inhibitor aprotinin is currently used to reduce blood loss and the need for blood transfusion after cardiopulmonary bypass. We have recently reported that aprotinin impairs endothelium-dependent but not endothelium-independent relaxations in rat thoracic aortic rings due to its inhibitory effect on endothelial nitric oxide (NO) production. In light of these findings, the current study was designed to investigate the effects of aprotinin on coronary endothelial function in isolated rat hearts and on the expression of endothelial NO synthase (eNOS) in cultured rat coronary microvascular endothelial cells (CMEC). METHODS: Hearts obtained from Sprague-Dawley rats were perfused on a constant flow Langendorff isolated heart system and coronary perfusion pressure and cardiac parameters were recorded. The coronary relaxant responses to bolus infusions of bradykinin (BK) and sodium nitroprusside (SNP) were recorded in the absence and presence of aprotinin. Total RNA and protein samples were extracted from CMEC incubated with aprotinin or the vehicle, 0.9% NaCl. RT-PCR-Southern blotting and Western analyses were carried out to assess eNOS mRNA and protein levels, respectively. RESULTS: Aprotinin (125 and 250 kIU/ml) increased coronary perfusion pressure without changing the heart rate and cardiac contractility. Aprotinin inhibited BK-induced coronary vasodilatation at 250 kIU/ml, but not at 125 kIU/ml concentrations. The relaxant response to SNP did not change in response to either concentration of the drug. Incubation of CMEC with aprotinin down-regulated eNOS mRNA and protein at 250 kIU/ml, but not at 125 kIU/ml concentration. CONCLUSION: These data suggest that aprotinin selectively inhibits NO synthesis at higher doses (> or = 250 kIU/ml) and therefore impairs endothelium-dependent coronary vascular tone. This effect of the drug may contribute to its 'blood-sparing' action, but may also account for the increase in the incidence of postoperative graft thrombosis observed in clinical practice during aprotinin therapy.     

白藜芦醇对心肌微血管内皮细胞缺血再灌注损伤的保护作用

目的观察白藜芦醇对缺血再灌注损伤心肌微血管内皮细胞(cardiac microvascular endothelial cells,CMECs)的保护作用。方法培养大鼠CMECs,模拟缺血再灌注损伤,随机分为对照组、缺血再灌注组及白藜芦醇组,采用四甲基偶氮唑盐微量酶反应比色法检测细胞活性;Annexin V-FITC/PI双标记流式细胞术检测CMECs凋亡率;酶联免疫吸附法检测半胱天冬酶3(caspase-3)活性、丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性。结果与对照组比较,缺血再灌注组和白藜芦醇组吸光度值明显降低(P<0.01);CMECs凋亡率、caspase-3相对活性、MDA含量、SOD活性明显升高,差异有统计学意义(P<0.05.P<0.01);与缺血再灌注组比较,白藜芦醇组吸光度值、SOD活性明显升高[(0.47±0.06)vs(0.14±0.04),(28.9±2.9)μU/L vs(22.4+2.2)μU/L,P<0.01];CMECs凋亡率、caspase-3相对活性、MDA含量明显降低.差异有统计学意义[(15.6±0.4)%vs(38.6±0.6)%,(152.1±13.3)vs(307.2±25.3),(9.2±0.7)μmol/L vs(13.5±1.2)μmol/L,P<0.01]。结论白藜芦醇可减轻CMECs的缺血再灌注损伤,这种保护作用可能是通过抗氧化和抗调亡来实现。     

血脂康对冠心病合并高脂血症患者血管内皮细胞的保护作用

目的　探讨血脂康调血脂治疗对冠心病合并高脂血症患者内皮细胞的保护作用。方法　 70例血浆胆固醇水平大于 5 .72mmol L并经冠状动脉造影证实的冠心病患者作为研究对象 ,4 8例给予降血脂治疗 ,2 2例给予安慰剂 ,另 10例无明显危险因素 ,年龄、性别相匹配的冠状动脉正常者作为对照。患病组分别于治疗前、后 4周及 8周采用高分辨率血管超声法检查上肢动脉内皮依赖性舒张功能 (VEDR) ,超速离心法检查血液中脱落的内皮细胞及放射免疫法测定血浆血友病因子 (vWf)的水平。结果　与正常对照组相比 ,患病组VEDR明显受损 ,脱落的内皮细胞也明显增加 ,血浆vWf的水平显著升高 (P <0 .0 1) ,治疗组 4周VEDR即有改善的趋势 ,8周后明显改善 (P <0 .0 5 ) ,而上肢动脉对硝酸甘油的反应性治疗前后未发生改变 ;治疗 8周后脱落的内皮细胞也明显减少 (P <0 .0 5 ) ,vWf的水平显著下降 (P <0 .0 1)。安慰剂组未见任何变化。结论　血脂康调血脂治疗可有效保护冠心病合并高脂血症患者的内皮细胞     

The effects of scleroderma serum on human microvascular endothelial cells

To investigate the vascular immunopathology of systemic sclerosis, we developed a model consisting of human microvascular endothelial cells, leukocytes, and serum. Sera from 19% of the patients studied mediated antibody-dependent cellular cytotoxicity against endothelial cells. Some sera also mediated cytotoxicity against aortic endothelium and fibroblasts. K lymphocytes, the cells that mediate antibody-dependent cellular cytotoxicity, were identified in the skin of some patients. The sera alone were not cytotoxic or growth inhibitory, and did not affect endothelial prostacyclin production.     

Effects of angiotensin II on nitric oxide generation in proliferating and quiescent rat coronary microvascular endothelial cells.

Nitric oxide (NO) and the mitogenic peptide angiotensin II (Ang II) have been implicated in endothelial cell growth. However, the putative relationship between these two opposing agents with respect to endothelial cell growth remains unknown. In this study, proliferating and confluent rat coronary microvascular endothelial cells (CMEC) were treated with different doses of Ang II, Ca2+ ionophore A23187, or valsartan (an Ang II type 1 (AT1) receptor inhibitor) alone or in combination for 24 h before measuring the nitrite levels as an index of NO generation. NO production and endothelial NO synthase (eNOS) mRNA/protein expression were found to be 3-fold greater in proliferating vs. quiescent CMEC. Treatments of CMEC with Ang II or Ca2+ ionophore A23187 equally increased NO production without altering the fold-difference in the basal release of NO from proliferating vs. confluent CMEC. Valsartan abolished NO production in CMEC treated with Ang II but not Ca2+ ionophore A23187. Treatments of endothelium-intact vascular rings with Ang II (1 nmol/l to 10 micromol/l) plus valsartan or PD-123319, an Ang II type 2 (AT2) receptor inhibitor, attenuated vascular responses to acetylcholine in an Ang II dose-dependent manner. In these rings, phenylephrine produced significant increases in contractile responses only at nmol/l concentrations of Ang II. In contrast, pharmacological and mechanical inactivation of endothelium enhanced contractile responses to phenylephrine at micromol/I concentrations of Ang II. These data demonstrate that Ang II stimulates NO production in CMEC in both an AT1- and an AT2 receptor-regulated manner, and that this stimulation of NO may be beneficial in counterbalancing the direct vasoconstrictor effect of Ang II on underlying smooth muscle cells.     

Des-acyl ghrelin protects microvascular endothelial cells from oxidative stress-induced apoptosis through sirtuin 1 signaling pathway

ObjectiveGhrelin is a stomach-derived hormone. Acylation of ghrelin has been essential for its biological activities such as stimulating appetite. On the other hand, the function of des-acyl ghrelin (Des-G) has not been fully elucidated. The aim of the present study is to examine the anti-apoptotic effect of Des-G on endothelial cells.Materials/MethodsAfter human retinal microvascular endothelial cells (RMECs) were pretreated with or without 100 nmol/L Des-G, apoptosis was induced with 0.1 mmol/L hydrogen peroxide (H₂O₂). For pharmacological inhibition of surtuin 1 (SIRT1) catalytic activity, the cells were treated with 10 μmol/L Ex-527. Inhibition of SIRT1 with siRNA was also performed. The quantitative estimation of DNA fragmentation was used as a marker of apoptosis. Furthermore, total SIRT activity in nuclear extracts, mRNA and protein levels of SIRT1, manganese superoxide dismutase (MnSOD) and catalase were determined.ResultsDes-G pretreatment protected RMECs from oxidative stress-induced apoptosis and increased SIRTs deacetylase activity in nuclear extracts. On the other hand, both pharmacological and siRNA mediated inhibition of SIRT1 attenuated the anti-apoptotic effect of Des-G. Moreover, Des-G increased mRNA and protein levels of SIRT1 and antioxidant enzymes such as MnSOD and CAT, which are downstream targets of SIRT1. Although the treatment of Ex-527 did not alter mRNA expression levels of SIRT1, it decreased mRNA expression levels of antioxidant enzymes in the cells with Des-G pretreatment.ConclusionsOur results suggest that SIRT1 signaling pathway contributes to protective effect of Des-G against oxidative stress-induced apoptosis.     

Basal nonselective cation permeability in rat cardiac microvascular endothelial cells

The presence of a basal nonselective cation permeability was mainly investigated in primary cultures of rat cardiac microvascular endothelial cells (CMEC) by applying both the patch-clamp technique and Fura-2 microfluorimetry. With low EGTA in the pipette solution, the resting membrane potential of CMEC was -21.2 +/- 1.1 mV, and a Ca(2+)-activated Cl(-) conductance was present. When the intracellular Ca(2+) was buffered with high EGTA, the membrane potential decreased to 5.5 +/- 1.2 mV. In this condition, full or partial substitution of external Na(+) by NMDG(+) proportionally reduced the inward component of the basal I-V relationship. This current was dependent on extracellular monovalent cations with a permeability sequence of K(+) > Cs(+) > Na(+) > Li(+) and was inhibited by Ca(2+), La(3+), Gd(3+), and amiloride. The K(+)/Na(+) permeability ratio, determined using the Goldman-Hodgkin-Katz equation, was 2.01. The outward component of the basal I-V relationship was reduced when intracellular K(+) was replaced by NMDG(+), but was not sensitive to substitution by Cs(+). Finally, microfluorimetric experiments indicated the existence of a basal Ca(2+) entry pathway, inhibited by La(3+) and Gd(3+). The basal nonselective cation permeability in CMEC could be involved both in the control of myocardial ionic homeostasis, according to the model of the blood-heart barrier, and in the modulation of Ca(2+)-dependent processes.