血管紧张素-(1-7)对血管紧张素II致人脐静脉内皮细胞损伤的保护作用研究

目的 :观察血管紧张素 (1 7) [Ang (1 7) ]对血管紧张素II(AngII)致人脐静脉内皮细胞 (HU VECs)损伤的保护作用。方法 :体外培养的HUVECs随机分为 4组 :对照组 ,AngII组 ,Ang (1 7)组 ,AngII Ang (1 7)组。采用分光光度计测定培养的HU VECs乳酸脱氢酶 (LDH)漏出 ;流式细胞仪检测细胞凋亡 ;硝酸还原酶法和放射免疫分析技术分别测定HUVECs上清液中一氧化氮 (NO)和内皮素 1(ET 1)的含量。结果 :与对照组比较 ,AngII(0 .1μmol·L-1)显著增加HUVECsLDH漏出 (P <0 .0 1)、ET 1分泌(P <0 .0 1)和HUVECs凋亡率 (P <0 .0 1) ,显著减少NO的含量 (P <0 .0 5 ) ;Ang (1 7)呈剂量依赖性抑制了AngII的促LDH漏出、ET 1分泌、增加细胞凋亡等作用 ,同时明显促进HUVECs的NO释放 ;单用Ang (1 7)对HUVECs无明显影响。结论 :Ang (1 7)可抑制AngII所致的体外培养HUVECs损伤 ,对内皮细胞具有保护作用。     

高糖对HUVEC功能的影响及机制研究

目的观察葡萄糖对人脐静脉内皮细胞(HUVECs)活性功能的影响及机制研究。方法内皮细胞培养液体外培养HUVEC,用不同浓度(10、20、30、40、50mmol/L)的葡萄糖作用于HUVEC,用NO含量、eNOS活性检测、细胞凋亡测定和细胞内ROS检测方法,观察不同浓度葡萄糖作用后的HUVEC功能活性的影响。结果高糖对HUVEC的增殖活性具有剂量依赖性抑制作用。高糖剂量依赖性抑制HUVEC细胞eNOs活性、NO的产生,增强细胞凋亡和细胞内ROS的生成。结论葡萄糖对人脐静脉内皮细胞(HUVECs)活性功能具有抑制作用,其机制与增强细胞凋亡和增加细胞内ROS有关。     

Genipin inhibits endothelial exocytosis via nitric oxide in cultured human umbilical vein endothelial cells

Aim： Exocytosis of endothelial Weibel-Palade bodies, which contain von Willebrand factor （VWF）, P-selectin and other modulators, plays an important role in both inflammation and thrombosis. The present study investigates whether genipin, an aglycon of geniposide, inhibits endothelial exocytosis. Methods： Human umbilical vein endothelial cells （HUVECs） were isolated from umbilical cords and cultured. The concentration of VWF in cell supernatants was measured using an ELISA Kit. P-selectin translocation on the cell surface was analyzed by cell surface ELISA. Cell viability was measured using a Cell Counting Kit-8. Mouse bleeding times were measured by amputating the tail tip. Western blot analysis was used to determine the amount of endothelial nitric oxide synthase （eNOS） and phospho-eNOS present. Nitric oxide （NO） was measured in the cell supernatants as nitrite using an NO Colorimetric Assay. Results： Genipin inhibited thrombin-induced VWF release and P-selectin translocation in HUVECs in a dose- and time- dependent manner. The drug had no cytotoxic effect on the cells at the same doses that were able to inhibit exocytosis. The functional study that demonstrated that genipin inhibited exocytosis in vivo also showed that genipin prolonged the mouse bleeding time. Furthermore, genipin activated eNOS phosphorylation, promoted enzyme activation and increased NO production. L-NAME, an inhibitor of NOS, reversed the inhibitory effects of genipin on endothelial exocytosis. Conclusion： Genipin inhibits endothelial exocytosis in HUVECs. The mechanism by which this compound inhibits exocytosis may be related to its ability to stimulate eNOS activation and NO production. Our findings suggest a novel antiinflammatory mechanism for genipin. This compound may represent a new treatment for inflammation and/or thrombosis in which excess endothelial exocytosis plays a pathophysiological role.     

淫羊藿苷对血管紧张素Ⅱ诱导人脐静脉内皮细胞损伤的保护作用

探讨淫羊藿苷对血管紧张素Ⅱ（AngⅡ）诱导损伤人脐静脉内皮细胞株（ECV-304）的影响。培养内皮细胞，淫羊藿苷作用24h后，采用AngII诱导制备内皮细胞损伤模型，测定细胞存活率（MTT法）、培养液中乳酸脱氢酶（LDH）释放量、NO值、清除超氧阴离子自由基（O2^-）和羟自由基（·OH）的能力，测定胞内超氧化物歧化酶（SOD）活性、T-NOS、iNOS以及eNOS的含量。与AngⅡ单独处理组相比，淫羊藿苷能明显提高细胞存活率，提高SOD、T-NOS和cNOS活力，提高NO含量，增强清除O2^-和·OH的能力，降低LDH和iNOS的量。结果表明淫羊藿苷对AngⅡ损伤的内皮细胞有一定的保护作用。     

非诺贝特对溶血卵磷脂诱导的血管内皮细胞增生及凋亡的影响

目的:观察非诺贝特对溶血卵磷脂(LPC)诱导的血管内皮细胞增生、凋亡的影响并探讨其机制。方法:体外培养人脐静脉内皮细胞(HUVECs),分为正常对照组、LPC组、非诺贝特低浓度组(10μmol·L-1)、非诺贝特中浓度组(50μmol·L-1)及非诺贝特高浓度组(100μmol·L-1)。分别观测LPC对血管内皮细胞增生、凋亡及凋亡调控蛋白抑制X连锁凋亡抑制蛋白(XIAP)的影响,及非诺贝特干预后的变化。结果:与正常对照组比较,LPC抑制内皮细胞增生,促进内皮细胞凋亡,XIAP表达减弱。非诺贝特可干预LPC对内皮细胞的作用,使内皮细胞增生增强,凋亡减少,XIAP表达增强。结论:非诺贝特可通过促进XIAP表达干预LPC对HUVECs增生及凋亡的影响。     

溶血卵磷脂对血管内皮细胞增殖及凋亡的影响

目的观测溶血卵磷脂(LPC)对血管内皮细胞增殖及凋亡的影响,从而探讨动脉硬化发生的机制。方法取体外培养的人脐静脉内皮细胞(HUVECs),在含不同浓度LPC(5、10、20mg/L)的培养基中分别培养6、12、24、48h,用四甲基偶氮唑蓝(MTT)比色法、流式细胞仪、荧光显微镜观测血管内皮细胞增殖及凋亡的变化。结果与正常对照组比较,LPC抑制内皮细胞增殖,促进细胞凋亡,且其作用呈时间-效应、浓度-效应依赖关系,但随着LPC浓度增加,细胞凋亡增加的同时细胞死亡比例也增加。结论LPC抑制血管内皮细胞增殖,促进细胞凋亡,可能是其致动脉粥样硬化机制之一。     

Angiotensin‐(1–7) treatment ameliorates angiotensin II‐induced apoptosis of human umbilical vein endothelial cells

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辛伐他汀对脐静脉内皮细胞金属基质蛋白酶9表达的影响

目的观察辛伐他汀对脐静脉内皮细胞（human umbilical vein endothelial cell,HUVEC）金属基质蛋白酶9（matrix metalloproteinase-9,MMP-9）表达的影响。方法采用逆转录聚合酶链反应及蛋白质免疫印迹分析检测MMP-9mRNA转录和蛋白水平表达,观察辛伐他汀不同浓度及不同孵育时间HUVECMMP-9表达的影响。结果辛伐他汀呈浓度和时间依赖性减低HU-VEC的MMP-9mRNA转录和蛋白水平的表达。结论辛伐他汀可抑制HUVE CMMP-9表达,防治动脉粥样硬化。     

苦参碱对U937细胞及人脐静脉血管内皮细胞增殖的影响

目的：观察苦参碱（Matrine，Mat）对U937细胞及人脐静脉血管内皮细胞（HUVECs）增殖的影响。方法：采用体外培养技术，通过细胞形态、MTT实验、细胞周期测定观察Mat对U937细胞及HUVECs增殖的影响。结果：Mat（0．2～0．5mg／mL）作用24h后对U937细胞均有增殖抑制作用（P〈0．05或P〈0．01），在该浓度范围内呈剂量和时间依赖性，其作用U937细胞48h的半数抑制浓度（IC50）约为0．4mg／mL，有效作用时间为1d；Mat（0．1～0．5mg／mL）作用24、48、72h后对HUVECs增殖无明显抑制作用，亦无剂量和时间依赖性（P〉0．05）；Mat（0．2～0．5mg／mL）作用U937细胞48h后，S期细胞比例增加，细胞发生S期阻滞（P〈0．05或P〈0．01）；Mat（0．1～0．5mg／mL）作用HUVECs 48h后，对细胞周期无明显影响（P〉0．05）。结论：一定浓度Mat对U937细胞具有增殖抑制作用，呈时间-剂量依赖关系。Mat在一定浓度和时间下不能抑制HUVECs增殖，对其细胞周期亦无明显影响。     

三氧化二砷对人脐静脉内皮细胞作用的研究

目的以人脐静脉内皮细胞作为血管新生内皮细胞的模拟物,通过研究As2O3对人脐静脉内皮细胞的影响,探讨As2O3的抗血管新生作用机制。方法分离和培养人脐静脉内皮细胞,以不同浓度和时间的As2O3和VEGF作用于脐静脉内皮细胞,应用流式细胞技术测定As2O3和VEGF对脐静脉内皮细胞增殖、细胞凋亡和癌基因bcl-2表达的影响。结果As2O3以时间和剂量依赖的方式抑制内皮细胞增殖,促进细胞凋亡,抑制癌基因bcl-2表达,而VEGF可拮抗As2O3的部分作用。结论As2O3可以通过直接作用于内皮细胞而抑制血管新生。     

阿魏酸钠对同型半胱氨酸致人脐静脉内皮细胞功能障碍的影响及机制

目的：采用人脐静脉内皮细胞建立了同型半胱氨酸血管内皮细胞损伤模型,研究阿魏酸钠对胞外同型半胱氨酸损伤的内皮细胞功能障碍的保护作用。方法：采用CCK8法检测不同浓度阿魏酸钠对人脐静脉内皮细胞活性的影响;人脐静脉内皮细胞与不同浓度阿魏酸钠和同型半胱氨酸共同孵育后,直接观察同型半胱氨酸对人脐静脉内皮细胞形态的影响,检测一氧化氮、一氧化氮合成酶、内皮素、丙二醛、超氧化物歧化酶、谷胱甘肽过氧化物酶含量变化及相应阿魏酸钠的保护作用。结果：阿魏酸钠能够减少同型半胱氨酸对人脐静脉内皮细胞的损伤,降低同型半胱氨酸对人脐静脉内皮细胞产生的氧化压力,减轻同型半胱氨酸诱导的人脐静脉内皮细胞分泌功能异常。结论：阿魏酸钠能够缓解同型半胱氨酸导致的人脐静脉内皮细胞功能障碍。     

Atorvastatin attenuates homocysteine-induced apoptosis in human umbilical vein endothelial cells via inhibiting NADPH oxidase-related oxidative stress-triggered p38MAPK signaling

Aim:

To examine the effect of atorvastatin on homocysteine (Hcy)-induced reactive oxygen species (ROS) production and apoptosis in human umbilical vein endothelial cells (HUVECs).

Methods:

HUVECs were cultured with Hcy (0.1−5 mmol/L) in the presence or absence of atorvastatin (1−100 μmol//L) or various stress signaling inhibitors, including the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium (DPI, 10 μmol/L), the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580 (10 μmol/L) and antioxidants N-acetyl cysteine (NAC, 1 mmol/L). Cell apoptosis was evaluated by Annexin V/propidium iodide staining and flow cytometry. ROS were detected by 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFH-DA). NADPH oxidases were evaluated with lucigenin-enhanced chemiluminescence. Hcy-induced expression of p38MAPK protein was measured by Western blotting analysis.

Results:

Atorvastatin inhibited endothelial cell apoptosis induced by 1 mmol/L Hcy in a dose-dependent manner and the maximal inhibitory effect was reached at 100 μmol/L. Atorvastatin (10 μmol/L) significantly suppressed Hcy (1 mmol/L for 30 min) induced ROS accumulation (3.17±0.33 vs 4.34±0.31, P<0.05). Atorvastatin (10 μmol/L) also antagonized Hcy (1 mmol/L for 30 min) induced activation of NADPH oxidase (2.57±0.49 vs 3.33±0.6, P<0.05). Furthermore, atorvastatin inhibited Hcy-induced phosphorylation of p38 MAPK (1.7±0.1 vs 2.22±0.25, P<0.05), similar effects occurred with DPI, NAC and SB203580.

Conclusion:

Atorvastatin may inhibit Hcy-induced ROS accumulation and endothelium cell apoptosis through an NADPH oxidase and/or p38MAPK-dependent mechanisms, all of which may contribute to atorvastatin-induced beneficial effect on endothelial function.     

糖化低密度脂蛋白介导的人脐静脉血管内皮细胞增殖及Toll样受体4表达变化与白藜芦醇干预后的影响

目的研究糖化低密度脂蛋白(glycated low density lipoprotein,Gly-LDL)对人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVEC)增殖及Toll样受体4(Toll-like receptor 4,TLR4)表达的影响和白藜芦醇干预后的变化。方法通过细胞技术试剂盒(cell counting kit-8,CCK-8)测定不同浓度Gly-LDL处理12、24及48 h后HUVEC增殖能力;实时定量聚合酶链式反应(quantitative real-time polymerase chain reaction,QRT-PCR)检测TLR4的表达量。分别采用CCK-8和QRT-PCR检测Gly-LDL加白藜芦醇干预24 h后HUVEC增殖能力及TLR4水平变化。结果不同浓度Gly-LDL干预12 h后高浓度组HUVEC细胞增殖较正常组下降,组内比较干预24 h、48 h后各浓度Gly-LDL组细胞增值率均较正常组下降,组间比较两者无统计学差异。与普通LDL组比较,TLR4信使 RNA表达水平随着干预时间和Gly-LDL浓度增加。不同浓度白藜芦醇干预24 h后,均能明显提高细胞增殖率,抑制Gly-LDL上调的TLR4信使RNA表达。结论 Gly-LDL可抑制内皮细胞增殖能力,并引起TLR4信号的激活。白藜芦醇能有效改善Gly-LDL介导的HUVEC增殖能力下降和TLR4信号水平的上调,减轻Gly-LDL对内皮细胞的损伤。     

脉络宁注射液对人血管内皮细胞缺氧损伤的保护作用

目的:研究脉络宁注射液对人脐静脉血管内皮细胞缺氧损伤的保护作用及其机制。方法:常规进行人血管内皮细胞(HUVECs)培养,将细胞随机分为正常对照组、缺氧组和脉络宁20 mg.L-1组。采用CCK-8法检测细胞存活率;Hochest33258荧光染料检测细胞凋亡率;RT-PCR法检测各组细胞中血管内皮生长因子(VEGF),血管生成素-2(Ang-2)mRNA表达水平。结果:脉络宁注射液可显著提高缺氧损伤细胞的存活率(P<0.05);与正常对照组比较,缺氧组细胞内VEGF和Ang-2 mRNA表达水平明显增高。与缺氧组比较,脉络宁组VEGF和Ang-2的表达进一步增强,各组间比较均有差异(P<0.05)。结论:脉络宁注射液可减轻血管内皮细胞缺氧损伤,上调缺氧血管内皮细胞中VEGF和Ang-2的表达,对血管内皮细胞缺氧损伤有一定的保护作用。     

Apoptosis of human umbilical vein endothelial cells induced by artesunate

Artesunate (ART), a semi-synthetic derivative of artemisinin isolated from the traditional Chinese herb Artemisia annua, is an effective novel antimalarial drug. The present study investigated the apoptotic activity of artesunate in cultured human umbilical vein endothelial cell (HUVEC) by means of nuclear staining, DNA agarose gel electrophoresis, and flow cytometry. The observations also indicated that artesunate induced apoptosis of HUVEC in a concentration-dependent and time-dependent manner. A Western immunoblot analysis showed down-regulation of the bcl-2 protein and up-regulation of the bax protein in the artesunate-treated HUVEC. Ca2+ in cells was evaluated by fluorescent spectrophotometer using Fura 2-AM as probe. These results suggest that artesunate may be a potential apoptosis-inducing agent for endothelial cells.     

Comparison of P25 and nanobelts on Kruppel-like factor-mediated nitric oxide pathways in human umbilical vein endothelial cells

Recently, we reported that titanium dioxide (TiO₂) materials activated endothelial cells via Kruppel-like factor (KLF)-mediated nitric oxide (NO) dysfunction, but the roles of physical properties of materials are not clear. In this study, we prepared nanobelts from P25 particles and compared their adverse effects to human umbilical vein endothelial cells (HUVECs). TiO₂ nanobelts had belt-like morphology but comparable surface areas as P25 particles. When applied to HUVECs, P25 particles or nanobelts did not induce cytotoxicity, although nanobelts were much more effective to increase intracellular Ti element concentrations compared the same amounts of P25 particles. Only nanobelts significantly induced THP-1 adhesion onto HUVECs. Consistently, nanobelts were more significant to induce the expression of intracellular adhesion molecule-1 (ICAM1) and the release of soluble ICAM-1 (sICAM-1), indicating that nanobelts were more potent to induce endothelial activation in vitro. As the mechanisms for endothelial activation, both P25 and nanobelts reduced the generation of intracellular NO as well as the expression of NO regulators KLF2 and KLF4. Combined, the results from this study indicated that the different morphologies of P25 particles and nanobelts only changed their internalization into HUVECs but showed minimal impact on KLF-mediated NO signaling pathways.     

微RNA-24对过氧化氢诱导的人脐静脉血管内皮细胞凋亡的影响及作用机制

目的探讨微RNA-24(miR-24)对H2O2诱导的人脐静脉血管内皮细胞(HUVEC)存活、迁移和凋亡的影响及作用机制。方法通过转染miR-24高表达、miR-24抑制物(anti-miR-24)及其阴性对照慢病毒质粒(miR-24 NC)构建稳转细胞,3种细胞用H2O2500μmol·L^-1处理12 h。采用四甲基偶氮唑盐(MTT)法检测细胞存活率,划痕实验检测细胞迁移率,Hoechst33258染色及流式细胞术检测细胞凋亡,实时荧光定量聚合酶链反应(qRT-PCR)检测miR-24,Bax,Bcl-2和胱天蛋白酶3 mRNA表达水平,Western印迹法和免疫细胞化学法检测Bax、Bcl-2和胱天蛋白酶3蛋白表达水平。结果与miR-24 NC组比,miR-24高表达组miR-24表达升高(P<0.05),而anti-miR-24组降低(P<0.05)。与正常对照组比,H2O2组细胞凋亡率增加(P<0.05)、细胞存活和迁移率降低(P<0.05),表明氧化损伤模型构建成功。与H2O2+miR-24 NC组比,H2O2+miR-24高表达组上述指标较H2O2+miR-24 NC组升高(P<0.05),促凋亡蛋白Bax和胱天蛋白酶3 mRNA与蛋白表达量增加,抑凋亡蛋白Bcl-2 mRNA和蛋白表达量降低(P<0.05),而H2O2+anti-miR-24组可降低细胞凋亡率、增加细胞存活和迁移率,降低Bax和胱天蛋白酶3 mRNA与蛋白表达水平,增加Bcl-2 mRNA和蛋白表达水平(P<0.05)。结论氧化应激状态下,miR-24可通过上调Bax、胱天蛋白3表达和下调Bcl-2蛋白表达,促进HUVEC凋亡和抑制其存活和迁移能力。     

小泛素相关修饰蛋白特异性蛋白酶1对脐静脉内皮细胞增殖、迁移和凋亡的影响

目的探讨小泛素相关修饰蛋白特异性蛋白酶1(SENP1)对脐静脉内皮细胞(HUVEC)增殖、迁移及凋亡的影响。方法用20个感染复数的p LKO.1-SENP1-小发夹RNA(shRNA)-绿色荧光蛋白(GFP)干涉慢病毒(SENP1干涉组)和p PIG-U6-随机序列(scramble)-GFP对照慢病毒(GFP-SC对照组)感染HUVEC。病毒感染HUVEC 48 h后,用流式细胞仪检测慢病毒的感染效率,实时荧光定量PCR(RT-PCR)和Western蛋白质印迹法检测HUVEC内SENP1基因和蛋白表达水平。用CCK8试剂盒检测细胞增殖能力,Transwell小室法检测细胞的迁移能力,流式细胞仪分析细胞凋亡率。结果 GFP-SC对照组和SENP1干涉组感染效率均>97%。与GFP-SC对照组比较,SENP1干涉组HUVEC SENP1基因和蛋白表达水平显著降低(P<0.01)。CCK8法测定结果表明,SENP1干涉组HUVEC增殖能力较GFP-SC对照组明显降低(P<0.01)。SENP1干涉组细胞迁移过Transwell小室的细胞数量明显低于GFP-SC对照组(P<0.01)。用低浓度血清(含2%胎牛血清)培养基诱导细胞凋亡24 h,SENP1干涉组细胞凋亡率即已升高到GFP-SC对照组的2倍左右(P<0.05);诱导凋亡至48 h,SENP1干涉组细胞凋亡率升高到对照组的3.2倍左右(P<0.05)。结论 HUVEC内SENP1表达水平的降低不仅可以显著抑制细胞增殖和迁移能力,亦可促进细胞凋亡。     

普纳替尼对人血管内皮细胞功能的影响

目的 观察普纳替尼(Ponatinib)对人血管内皮细胞的增殖、迁移、血管形成及NO合成释放的影响, 从细胞水平评价Ponatinib血栓不良反应形成机制。方法 采用CCK-8法、体外划痕法、Matrigel基底膜成管法及NO检测试剂盒分别考察Ponatinib对人脐静脉内皮细胞(HUVECs)的细胞毒作用、迁移能力、血管形成能力及细胞NO释放水平的影响。结果 研究表明Ponatinib对HUVECs细胞半数抑制浓度(IC₅₀)为(0.69±0.05)μmol/L。非毒性剂量的Ponatinib能够不同程度抑制HUVECs细胞的迁移、血管形成及NO的释放(P<0.05)。结论 Ponatinib通过影响人血管内皮细胞增殖、迁移、血管形成等正常功能而造成血管内皮损伤, 可能为其诱发血栓的原因之一。