牙龈卟啉单胞菌对脐静脉血管内皮细胞表达IL-8和MCP-1 mRNA的影响

目的：在转录水平上观察牙龈卟啉单胞菌对脐静脉血管内皮细胞（human umbilical vein endothelial cells,HUVECs）表达白细胞介素-8（IL-8）和单核细胞趋化蛋白-1（MCP-1）的影响。方法：厌氧培养Pg，原代培养HUVECs，RNA抽提，逆转录-聚合酶链式反应（RT-PCR）和mRNA比色定量法检测IL-8和MCP-1基因表达。结果：HUVECs基础表达IL-8和MCP-1mRNA,Pg以剂量依赖的方式增强IL-8和MCP-1mRNA的表达，Pg感染后1hIL-8mRNA开始增高，3h达到高峰，并持续到5h。而MCP-1的mRNA从Pg感染后1h开始增高，3h达到高峰，5h出现下降趋势。结论：Pg在转录水平上增强UHUVECs表达IL-8和MCP-1，这种上调作用可能是牙周病早期基因水平调控炎症细胞募集的重要因素，可能是牙周疾病的炎症反应和免疫反应中主要调控机制之一。     

参芎葡萄糖注射液对H2O2诱导的HUVEC细胞氧化损伤的保护作用

目的:探讨参芎葡萄糖注射液(Shenxiong glucose injection,SGI)对H_2O_2诱导的人脐静脉上皮细胞(HUVEC)细胞氧化模型损伤的保护作用及其机制。方法:体外培养HUVEC人脐静脉内皮细胞,用H_2O_2(130 mmol·L~(-1))处理0.5 h,建立HUVEC细胞H_2O_2氧化损伤模型。SGI组HUVEC细胞用(6%,8%,10%)SGI预处理6 h后,再用H_2O_2处理0.5 h。用MTS法检测细胞存活率;用ELISA法检测乳酸脱氢酶(LDH)漏出量、丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-Px)活力;用实时荧光定量聚合酶链式反应(Real-time PCR)和蛋白质免疫印迹(Western blot)技术检测凋亡相关基因及蛋白B细胞淋巴瘤/白血病-2(Bcl-2),Bcl-2相关X蛋白(Bax)和半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)的mRNA和蛋白表达情况。结果:在130 mmol·L~(-1)H_2O_2作用细胞0.5 h的情况下,细胞存活率降低至50%左右,降低的程度合适,且实验结果重复性好,因此后续实验用该条件建立氧化损伤模型。与H_2O_2组比较,SGI预处理6 h能显著升高细胞存活率(P0.05,P0.01),减少LDH的外漏和MDA的生成(P0.05,P0.01),显著增加SOD,GSH-Px和CAT的活性(P0.05,P0.01)。RT-PCR和Western blot结果表明,SGI能显著上调Bcl-2的表达(P0.05,P0.01),下调Caspase-3,Bax的表达(P0.05,P0.01)。结论:参芎葡萄糖注射液能保护HUVEC细胞对抗H_2O_2诱导的氧化损伤,具有一定的剂量依赖关系,其作用机制可能与抑制细胞凋亡有关。     

复方银杏叶颗粒改善人脐静脉内皮细胞氧化应激损伤的作用及其机制研究

该实验于体外使用人脐静脉内皮细胞(HUVEC)与复方银杏叶颗粒(80,160,320,640 mg·L-1)预孵育,合并使用H2O2(1 200μmol·L-1)建立氧化应激损伤模型。采用MTT法研究药物对HUVEC细胞增殖情况的影响,检测细胞培养上清中的LDH,MDA,NO含量及SOD活性,判断药物对内皮细胞的保护作用。通过Casepase-3活性检测以及Annexin V-FITC/PI流式细胞术,检测药物对细胞凋亡的保护作用。使用Western blot法测定凋亡相关蛋白Bcl-2和Bax的表达。研究发现1 200μmol·L-1H2O2能够诱导内皮细胞产生氧化应激损伤,使细胞存活率降低,且细胞增殖抑制程度与H2O2的作用时间呈正相关。而80,160,320,640 mg·L-1的复方银杏叶颗粒能够明显减轻该模型下内皮细胞的氧化应激损伤,恢复细胞正常增殖水平,改善细胞状态,抑制细胞凋亡,同时能够上调Bcl-2的表达以及下调Bax的蛋白表达。该实验证明了复方银杏叶颗粒对H2O2诱导的内皮细胞氧化应激损伤及细胞凋亡具有一定的保护作用,其机制可能与复方银杏叶颗粒抑制了内皮细胞凋亡的线粒体途径有关。     

RNA干扰技术下调CEACAMl基因表达对胶质瘤血管生成的影响

目的：利用RNA干扰技术下调胶质瘤细胞中癌胚抗原相关细胞黏附分子1（ CEACAM1）基因的表达情况,探讨胶质瘤细胞CEACAM1表达变化后其培养上清液诱导人脐静脉内皮细胞（ HUVEC）增殖、血管生成的情况及其可能机制。方法设计并化学合成针对CEACAM1的3对siRNA,脂质体法瞬时转染SHG44细胞,收集细胞培养上清液作为条件培养基。分别采用RT－PCR检测RNA干扰后细胞中CEACAM1及血管内皮生长因子（ VEGF） mRNA表达的变化情况,ELISA法检测上清液中VEGF蛋白的含量。 MTT比色法及Transwell侵袭实验检测共培养刺激后HUVEC增殖及迁移能力的变化,体外血管生成实验检测体外血管生成能力。结果与正常对照组和阴性对照组相比,转染CEACAM1－siRNA后胶质瘤细胞中CEACAM1 mRNA的表达水平下调（P＜0．01）,以CEACAM1－siRNA3组最为显著。 RNA干扰后SHG44细胞VEGF mRNA及上清液中的VEGF蛋白含量均减少,HUVEC的增殖受到抑制,迁移及体外血管生成能力下降。结论 CEACAM1－siRNA能够下调SHG44细胞中CEACAM1 mRNA的表达,并通过减少VEGF分泌,从而影响HUVEC的增殖、迁移和体外血管生成能力。     

Differential Effects of Fondaparinux and Bemiparin on Angiogenic and Vasculogenesis-like processes

Introduction

Conventional therapy for venous thromboembolism or acute coronary syndrome involves the administration of glycoanticoagulants (heparins) or oligosaccharides (fondaparinux). We evaluated the effects of such drugs on angiogenesis and vasculogenesis-like models.

Materials and Methods

Human umbilical vein endothelial cells or human endothelial progenitor cells were treated with bemiparin, fondaparinux or unfractionated heparin, at concentrations reflecting the doses used in clinical practice. After 24 h, cell viability, proliferation, tubule formation and angiogenic molecular mechanisms, such as activation of the serine/threonine kinase AKT, were assessed. In vivo angiogenesis was studied using a Matrigel sponge assay in mice.

Results

Bemiparin gave a significant decrease of in vitro angiogenesis as shown by the reduction of endothelial cell tubule network, while both fondaparinux and unfractionated heparin did not show any significant effect. In assays of Matrigel sponge invasion in mice, unfractionated heparin was able to stimulate angiogenesis and, conversely, bemiparin inhibited angiogenesis. Furthermore, both bemiparin and fondaparinux caused a significant reduction in an in vitro vasculogenesis-like model, as demonstrated by the decrease of tubule network after co-seeding of endothelial progenitor cells and human umbilical vein endothelial cells. In addition, unfractionated heparin but not bemiparin was able to increase AKT phosphorylation.

Conclusions

In in vitro experiments, bemiparin was the only drug to show an anti-angiogenic and vasculogenic-like effect, unfractionated heparin showed only a trend to increase in angiogenesis assay and fondaparinux affected only the vasculogenesis-like model. Notably, the in vivo experiments corroborated these data. Such results are important for the choice of a patient-tailored therapy.     

Protein phosphatase 2B inhibition promotes the secretion of von Willebrand factor from endothelial cells

Background:  Secretion of Weibel–Palade body (WPB) contents is regulated, in part, by the phosphorylation of proteins that constitute the endothelial exocytotic machinery. In comparison to protein kinases, a role for protein phosphatases in regulating endothelial exocytosis is undefined. Objective and method:  In this study, we investigated the role of protein phosphatase 2B (PP2B) in the process of endothelial exocytosis using pharmacological and gene knockdown approaches. Results:  We show that inhibition of protein phosphatase 2B (PP2B) activity by cyclosporine A (CsA), tacrolimus or a cell-permeable PP2B autoinhibitory peptide promotes the secretion of ultralarge von Willebrand factor (ULVWF) from human umbilical vein endothelial cells (HUVECs) in the absence of any other endothelial cell-stimulating agent. PP2B inhibitor-induced secretion and anchorage of ULVWF strings from HUVECs mediate platelet tethering. In support of a role for PP2B in von Willebrand factor (VWF) secretion, the catalytic subunit of PP2B interacts with the vesicle trafficking protein, Munc18c. Serine phosphorylation of Munc18c, which promotes granule exocytosis in other secretory cells, is increased in CsA-treated HUVECs, suggesting that this process may be involved in CsA-mediated WPB exocytosis. Furthermore, the plasma VWF antigen level is also enhanced in CsA-treated mice, and small interfering RNA-mediated knockdown of the α and β isoforms of the PP2B-A subunit in HUVECs enhanced VWF secretion. Conclusions:  These observations suggest that CsA promotes VWF release, in part by inhibition of PP2B activity, and are compatible with the clinically observed association of CsA treatment and increased plasma VWF levels in humans.     

Anti-angiogenic action of 5,5-diphenyl-2-thiohydantoin-N10 (DPTH-N10)

Previously, we demonstrated that 5,5-diphenyl-2-thiohydantoin (DPTH) exerts an anti-proliferation effect on subcultured human umbilical vein endothelial cells (HUVEC). In the present study, we show that 2(naphthalen-2-ylmethylsulfanyl)-5,5-diphenyl-1,5-dihydro-imidazol-4-one (DPTH-N10), a derivative compound of DPTH, exerts a 5 times stronger inhibition of [3H]thymidine incorporation into HUVEC as compared with DPTH and at very low concentrations (0-20 microM) inhibited DNA synthesis and decreased cell number in cultured HUVEC in a concentration- and time-dependent manner, but not in human fibroblasts. [3H]thymidine incorporation analysis demonstrated that treatment of HUVEC with DPTH-N10 arrested the cell at the G0/G1 phase of the cell cycle. Western blot analysis revealed that the protein level of p21 in HUVEC increased after DPTH-N10 treatment. In contrast, the protein levels of p27, p53, cyclins A, D1, D3 and E, cyclin-dependent kinase (CDK)2, and CDK4 in HUVEC were not changed significantly after DPTH-N10 treatment. Immunoprecipitation showed that the formation of the CDK2-p21 complex, but not the CDK2-p27, CDK4-p21, and CDK4-p27 complex, was increased in the DPTH-N10-treated HUVEC. Kinase assay further demonstrated that CDK2, but not CDK4, kinase activity was decreased in the DPTH-N10-treated HUVEC. Pretreatment of HUVEC with a p21, but not p27, antisense oligonucleotide reversed the DPTH-N10-induced inhibition of [3H]thymidine incorporation into HUVEC. Taken together, these data suggest that DPTH-N10 inhibits HUVEC proliferation by increasing the level of p21 protein, which in turn inhibits CDK2 kinase activity, and finally interrupts the cell cycle. Capillary-like tube formation, aortic ring culture, and chick embryo chorioallantoic membrane (CAM) assays further demonstrated the anti-angiogenic effect of DPTH-N10.     

黄连解毒汤含药血清对LPSTNF-α诱导的人中性粒细胞与血管内皮细胞间粘附的影响

以体外培养的人脐静脉内皮细胞（HUVEC）为模型,应用蛋白染料染色法,观察HLJDT含药血清及其与LPS／TNF－α共同处理HUVEC4h、12h、24h后,对中性粒细胞与血管内皮细胞粘附的影响。结果显示,HLJDT含药血清不仅能抑制非致炎状态下中性粒细胞与血管内皮细胞的粘附,而且能抑制致炎因子所诱导的中性粒细胞与血管内皮细胞粘附作用增强,这可能是黄连解毒汤抗炎作用的机制之一。