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目的观察miR-124对内皮细胞增殖和凋亡的影响,探讨miR-124在动脉粥样硬化中的作用。方法采用荧光定量PCR检测miR-124在40例冠心病患者及40例正常对照血浆中的表达水平,CCK-8和BrdU法检测miR-124对人脐静脉内皮细胞（HUVECs）增殖的影响,TUNNEL染色检测miR-124对HUVECs凋亡的影响,Western blotting检测miR-124对HUVECs促凋亡蛋白cleaved caspase 3及抗凋亡蛋白Bcl-2表达的影响。结果与对照组比较,miR-124在冠心病患者血浆中的表达明显降低。CCK-8和BrdU检测结果提示miR-124可明显促进HUVECs增殖;TUNNEL染色及Western blotting检测结果提示,miR-124可明显降低HUVECs凋亡细胞的比例及cleaved caspase 3表达,同时增加Bcl-2的表达。结论Mi R-124可促进HUVECs的增殖并抑制其凋亡,为深入认识动脉粥样硬化的发病机制提供了理论依据。

目的 为制备血管化胰岛,分离、培养脂肪来源干细胞（adipose derived stem cells,ADSCs）,观察细胞共培养条件下,ADSCs对人脐静脉内皮细胞（human umbilical vein endothelial cell,HUVECs）增殖及成血管化功能的促进作用并探讨其机制。方法 采用胶原酶消化法分别原代培养获得ADSCs与HUVECs,细胞形态学、免疫荧光或多向诱导分化鉴定,建立HUVECs与ADSCs接触式及间接共培养体系,设立HUVECs单独培养为对照组,比较两组成血管化功能、HUVECs增殖状况及上清液血管内皮生长因子（vascular endothelial growth factor,VEGF）、碱性成纤维生长因子（basic fibroblast growth factor,b-FGF）浓度。结果 通过原代培养成功获得ADSCs与HUVECs;第3代ADSCs呈均一的长梭形纤维细胞样形态,免疫荧光检测见CD44/CD49d（＋）、CD31/CD34（-）,并具有多向分化功能;第2代HUVECs免疫荧光检测示vWF/CD31（＋）。于Matrigel内接触式共培养4h,ADSCs＋HUVECs组血管密度高于HUVECs组;间接共培养时,HUVECs生长曲线于ADSCs＋HUVECs组上移,在对数生长期的第3、4、5天,ADSCs＋HUVECs组HUVECs计数为（4.52±0.31）×10^4、（7.18±0.45）×10^4、（8.23±0.36）×10^4,大于单独HUVECs组的（2.71±0.25）×104、（4.87±0.26）×10^4、（6.86±0.33）×10^4（P〈0.01）;ADSCs＋HUVECs组HUVECs群体倍增时间为（1.36±0.23）d,短于单独HUVECs组的（1.62±0.31）d。四甲基噻唑蓝（methylthiazol tetraztlium,MTT）法测定HUVECs的A值培养第1、3、5、7天的ADSCs＋HUVECs组高于单独HUVECs组（P〈0.01）。培养第3、7、13天时ADSCs＋HUVECs组上清液VEGF、b-FGF浓度均高于HUVECs组（P〈0.01）。结论 ADSCs与HUVECs共培养时,ADSCs可能通过分泌或增加HUVECs分泌VEGF、b-FGF等细胞因子,进而促进HUVECs增殖及成血管化。

目的观察p54nrb基因沉默对人脐静脉内皮细胞（HU VEC）增殖、迁移及体外血管生成的影响,探讨p54nrb基因在血管新生中作用。方法用含有靶向人p54nrb基因sh RNA的慢病毒感染HUVEC 24h后,用嘌呤霉素进行筛选,Western blotting检测HUVEC中p54nrb蛋白的表达,确定p54nrb基因沉默效率,建立稳定的p54nrb基因沉默的HUVEC细胞系。CCK-8法检测p54nrb基因沉默对HUVEC细胞增殖的影响;划痕实验和Transwell迁移实验检测p54nrb基因沉默对HUVEC细胞迁移的影响;体外血管生成实验检测p54nrb基因沉默对HUVEC体外血管生成能力的影响。结果Western blotting结果显示,p54nrb基因沉默的HUVEC细胞中p54nrb蛋白表达显著减少,表明建立了稳定的p54nrb基因沉默的HUVEC细胞系。CCK-8实验结果显示,p54nrb基因沉默轻度地促进了HUVEC细胞增殖;划痕实验和Transwell迁移实验结果显示,p54nrb基因沉默后HUVEC细胞迁移能力明显下降;体外血管生成实验结果显示,p54nrb基因沉默明显抑制了HUVEC的体外血管生成能力。结论 p54nrb具有促进HUVEC细胞迁移及体外血管生成的作用,可能是一个新的参与血管生成的调控蛋白。

目的探讨内质网应激（ERS）在尿毒症血清致内皮细胞功能异常中的作用。方法采集尿毒症患者和正常健康人的静脉血血清。以不同浓度的尿毒症血清或正常血清（2.5%、5%、10%、15%、20%）刺激人脐静脉内皮细胞（hUVEC）24h后,采用MTT比色法检测细胞活力的变化,流式细胞术检测细胞周期,Hoechst33342染色观察细胞凋亡,荧光定量RT-PCR法检测单核细胞趋化蛋白-1（MCP-1）和葡萄糖调节蛋白78（GRP78）mRNA表达的变化。结果当浓度小于10%时,尿毒症血清对hUVEC细胞增殖率和S期百分比的影响随浓度增加而增大。当浓度大于10%时,hUVEC细胞增殖率和S期百分比逐渐下降,且细胞凋亡率逐渐增高。与正常血清组比较,10%尿毒症血清刺激后hUVEC GRP78和MCP-1 mRNA表达均显著升高（P〈0.05）。应用分子伴侣4-苯基丁酸（4-PBA）干预,可显著抑制尿毒症血清诱导的GRP78 mRNA和MCP-1 mRNA表达（P〈0.05）,降低hUVEC的增殖率和S期百分比（P〈0.05）。结论内质网应激可能是尿毒症血清诱导内皮细胞功能异常的重要细胞内机制。

背景与目的血管发生、新血管的形成是肿瘤生长及其转移灶形成的根本原因，因此，抗血管生成治疗已成为目前一种极具潜能的肿瘤治疗策略。本实验利用鸡胚胎模型和人脐静脉内皮细胞（Human umbilical vein endothelial cells，HUVECs）对牡蛎多肽（Oyster polypeptide,OPP）在体内及体外的抗血管生成作用及其机制进行了研究。方法利用鸡胚胎绒毛尿囊膜血管发育研究体内OPP对血管形成作用；MTT、平板划痕、Transwell板、管腔形成及电镜等方法观察OPP对HUVECs的增殖、迁移、侵袭等作用。结果体内实验表明0PP能明显抑制鸡胚胎绒毛尿囊膜血管的形成。MTT结果表明OPP能明显抑制HUVECs的增殖，IC50为400μg／mL。平板划痕法结果表明，200μg／mL、400μg／mL和800μg／mLOPP作用12h对HUVECs迁移的抑制率分别为18．75％、37．93％和74．07％；侵蚀实验结果显示200μg／mL、4008g／mL和800μg／mL的OPP对HUVECs迁移的抑制率分别为15．5％、37．29％和67．24％（P〈0．05）。管腔形成结果表明200μg／mL、400μg／mL和800μg／mL的OPP对HUVECs（作用10h）小管形成的抑制率分别为52．43％、84．47％和96．12％（P〈0．01）。此外，电镜结果显示，400μg／mL的OPP对HUVECs作用48h能诱导细胞明显凋亡。结论OPP可通过抑制内皮细胞增殖、迁移、管腔形成和促进内皮细胞凋亡，而具有显著抗血管生成作用。

目的研究非洛地平（felodipine）对氧化型低密度脂蛋白（ox-LDL）损伤的人脐静脉内皮细胞（HUVECs）活性氧（ROS）及细胞间粘附分子-1（ICAM-1）、血管细胞粘附分子-1（VCAM-1）mRNA表达的影响,探讨其独立于降血压外的抗动脉粥样硬化的作用机制。方法筛选ox-LDL与HUVECs细胞孵育最适的干预浓度梯度与时间,然后与不同浓度的非洛地平（0.1、1、10μmol/L）共同孵育24h,流式细胞仪测定细胞内ROS,realtime-PCR检测细胞ICAM-1、VCAM-1 mRNA的表达。结果 ox-LDL对HUVECs内ROS的产生呈浓度依赖效应,随着刺激浓度的升高,ROS产生增多,25mg/Lox-LDL作用最为显著（P〈0.01）,非洛地平可抑制ox-LDL诱导的HUVECsROS（P〈0.05）的产生;随ox-LDL刺激浓度的增加,ICAM-1、VCAM-1 mRNA表达逐渐上调,当浓度达到25mg/L时,作用最为显著（P〈0.05）,非洛地平可下调ICAM-1、VCAM-1 mRNA的表达（P〈0.05）。结论非洛地平可抑制ox-LDL诱导的HUVECs ROS的产生以及下调ICAM-1、VCAM-1 mRNA表达,发挥抗氧化抗炎的内皮保护功能。

Aim： To investigate the effects of glucagon-like peptide-1 （GLP-1） on endothelial NO synthase （eNOS） in human umbilical vein endothelial cells （HUVECs）, and elucidate whether GLP-1 receptor （GLP-1R） and GLP-1（9-36） are involved in these effects. Methods： HUVECs were used. The activity of eNOS was measured with NOS assay kit. Phosphorylated and total eNOS proteins were detected using Western blot analysis. The level of eNOS mRNA was quantified with real-time RT-PCR. Results： Incubation of HUVECs with GLP-1 （50-5000 pmol/L） for 30 min significantly increased the activity of eNOS. Incubation of HUVECs with GLP-1 （500-5000 pmol/L） for 5 or 10 min increased eNOS phosphorytated at ser-1177. Incubation with GLP-1 （5000 pmol/L） for 48 h elevated the level of eNOS protein, did not affect the level of eNOS mRNA. GLP-1R agonists exenatide and GLP-1（9-36） at the concentration of 5000 pmol/L increased the activity, phosphorylation and protein level of eNOS. GLP-1R antagonist exendin（9-39） or DPP-4 inhibitor sitagliptin, which abolished GLP-1（9-36） formation, at the concentration of 5000 pmol/L partially blocked the effects of GLP-1 on eNOS. Conclusion： GLP-1 upregulated the activity and protein expression of eNOS in HUVECs through the GLP-1R-dependent and GLP-1（9-36）-related pathways. GLP-1 may prevent or delay the formation of atherosclerosis in diabetes mellitus by improving the function of eNOS.

Sites of implantation with compromised biology may be unable to achieve the required level of angiogenic and osteogenic regeneration. The specific function and contribution of different cell types to the formation of prevascularized, osteogenic networks in co-culture remains unclear. To determine how bone marrow-derived mesenchymal stromal cells （BMSCs） and endothelial cells （ECs） contribute to cellular proangiogenic differentiation, we analysed the differentiation of BMSCs and ECs in standardized monolayer, Transwell and co-cultures. BMSCs were derived from the iliac bone marrow of five patients, characterized and differentiated in standardized monolayers, permeable Transwells and co-cultures with human umbilical vein ECs （HUVECs）. The expression levels of CD31, von Willebrand factor, osteonectin （ON） and Runx2 were assessed by quantitative reverse transcriptase polymerase chain reaction. The protein expression of alkaline phosphatase, ON and CD31 was demonstrated via histochemical and immunofluorescence analysis. The results showed that BMSCs and HUVECs were able to retain their lineage-specific osteogenic and angiogenic differentiation in direct and indirect co-cultures. In addition, BMSCs demonstrated a supportive expression of angiogenic function in co-culture, while HUVEC was able to improve the expression of osteogenic marker molecules in BMSCs.

Somatic cells can be reprogrammed into induced pluripotent stem cells （iPSCs） by defined factors. The low efficiency of reprogramming and genomic integration of oncogenes and viral vectors limited the potential application of iPSCs. Here we report that Lithium （Li）, a drug used to treat mood disorders, greatly enhances iPSC generation from both mouse embryonic fibroblast and human umbilical vein endothelial cells. Li facilitates iPSC generation with one （Oct4） or two factors （OS or OK）. The effect of Li on promoting reprogramming only partially depends on its major target GSK3β Unlike other GSK3β inhibitors, Li not only increases the expression of Nanog, but also enhances the transcriptional activity of Nanog. We also found that Li exerts its effect by promoting epigenetic modifications via downregulation of LSD1, a H3K4-specific histone demethylase. Knocking down LSD1 partially mimics Li＇s effect in enhancing reprogramming. Our results not only provide a straightforward method to improve the iPSC generation efficiency, but also identified a histone demethylase as a critical modulator for somatic cell reprogramming.

Aim： Chemokines usually direct the movement of circulating leukocytes to sites of inflammation or injury. CXCL1/GRO-a has been shown to be upregulated in atherosclerotic lesions and various cancers. The aim of this study was to investigate the mechanisms underlying the TNF-α-induced release of CXCL1 from human vascular endothelial cells in vitro. Methods： Human umbilical vein endothelial cells （HUVECs） were treated with different proinflam-matory mediators and growth factors. CXCL1 expression and secretion were determined using RT-PCR and ELISA, respectively. TNF-a-induced cell signaling was assayed with Western blotting. Cell viability/growth was determined using MTTassay. Monocyte migration was measured with transwell migration assay. Results： Among the 17 mediators and growth factors tested, TNF-α, LPS and thrombin induced marked increase in CXCL1 release from HUVEC cells. TNF-α （2, 5 ng/mL） induced CXCL1 release and mRNA expression in the cells in concentration- and time-dependent manners. TNF-α （5 ng/mL） caused activation of JNK, p38 MAPK, PI3K and Akt, whereas pretreatment with JNK inhibitor （SP600125）, p38 MAPK inhibitor （SB202190） or PI-3K inhibitor （LY294002） significantly suppressed TNF-a-induced CXCL1 release from the cells. But only SP600125 significantly reduced TNF-a-induced CXCL1 mRNA expression in the cells. Moreover, dexamethasone （up to 500 nmol/L） failed to affect TNF-a-induced CXCL1 release from the cells. In functional studies, recombinant CXCL1 enhanced HUVEC proliferation, and both recombinant CXCL1 and TNF-a-induced CXCL1 from HUVECs attracted human monocyte migration. Conclusion： TNF-a stimulates CXCL1 release from human ECs through JNK-mediated CXCL1 mRNA expression and p38 MAPK- and PI-3K-mediated CXCL1 secretory processes.