白花丹参对过氧化氢致人脐静脉血管内皮细胞损伤的保护作用

目的研究白花丹参对过氧化氢（H2O2）所致人脐静脉血管内皮细胞（HUVEC）损失的保护作用。方法应用酶消化灌注法培养人脐静脉血管内皮细胞,采用形态学观察和Ⅷ因子抗体免疫荧光检测法进行鉴定;取对数生长期的细胞分组进行干预,应用形态学观察法和MTT法检测各组血管内皮细胞的活性。结果H2O2损伤模型组细胞损伤明显,经白花丹参高、中、低各剂量组的干预,受损情况均有较明显地改善,使细胞活性（OD值）增高。结论白花丹参对H2O2所致的HUVEC损伤具有保护作用。     

丹参素对CD11b、P-selectin、ICAM-1、VCAM-1、E-selectin表达的影响

目的　考察丹参素对血小板、白细胞和血管内皮细胞表达细胞粘附分子CD11b、P selectin、ICAM 1、VCAM 1、E selectin的影响 ,以期探明其抗血栓形成作用的机制。方法　用流式细胞仪测定由TNFα、fMLP和凝血酶诱导细胞表面细胞粘附分子的表达。结果　人粒细胞受到fMLP刺激后 ,细胞表面CD11b表达明显增加。丹参素与粒细胞预孵30min后 ,则剂量依赖性地抑制CD11b的表达。血小板与凝血酶 (1× 10 3U·L-1)共孵 2 0min ,可明显增加血小板表面P selectin的表达。丹参素 (10 0～ 30 0mg·L-1)与血小板预孵 30min ,对凝血酶诱导的血小板表面P selectin的表达未见明显抑制作用。人脐静脉内皮细胞 (HUVEC)经TNFα处理后 ,明显增加细胞表面ICAM 1、VCAM 1和E selectin的表达 ,不同浓度的丹参素与HUVEC预孵 2h ,对TNFα诱导HUVEC表面ICAM 1的表达未见明显抑制作用 ,对TNFα诱导的VCAM 1、E selectin表达则可产生明显的抑制作用。结论　丹参素可抑制血管内皮细胞和粒细胞表达细胞粘附分子。这可能是丹参素发挥抗血栓形成作用的机制之一。     

七叶皂苷钠致静脉损伤作用细胞水平机制研究

目的了解七叶皂苷钠（Esc Na）对人脐静脉内皮细胞（HUVEC）活性及细胞周期分布的影响。方法在体外原代培养的HUVEC中加入不同浓度Esc Na及肿瘤坏死因子（TNF α）,孵育24 h后,采用MTT和中性红法对HUVEC活性进行定量分析,流式细胞技术对细胞周期进行分析。结果MTT法和中性红法显示Esc Na的半数抑制浓度（IC50）为（42.89±3.42）和（44.19±3.58） μg•mL 1。10～80 μg•mL 1浓度范围内的Esc Na可不同程度地抑制HUVEC生长,呈浓度依赖性。细胞周期分析结果也显示,Esc Na可阻抑G0/G1期细胞向S期的转变,抑制HUVEC增殖。结论七叶皂苷钠通过降低HUVEC活性,抑制细胞增殖,从而对HUVEC造成损伤。     

雌激素功能性调节人内皮细胞的生长调节致癌基因α表达

目的：研究雌激素在体外调节人脐带静脉内皮细胞（HUVEC）的生长调节致癌基因α（GROα）。方法：以体外培养的HUVEC为模型,Northern法检测CXC族趋化因子GROα mRNA;ELISA方法检测细胞表面的GROα蛋白表达;静态细胞粘附实验测定细胞表面的GROα蛋白的生理意义。结果：17β－雌二醇（0．05μmol/L）明显抑制HUVEC产生GROα mRNA和蛋白表达水平;而且17β－雌二醇抑制其蛋白表达水平显示剂量依赖性关系;雌激素受体α拮抗剂他莫昔芬（0．1μmol/L）单独使用不影响其蛋白表达,但可显著逆转17β－雌二醇抑制的GROα蛋白表达,显著逆转17β－雌二醇抑制单核细胞系细胞U937细胞粘附的HUVEC的作用达。结论：通过内皮细胞上雌激素受体α,雌激素可能功能性调节人内皮细胞的GROα的表达。     

芹菜素延缓人脐静脉内皮细胞衰老的体外实验研究

目的：探究芹菜素对人脐静脉内皮细胞衰老的延缓作用及相关机制。方法：将人脐静脉内皮细胞培养至第12代（老年细胞）,且自第4代（幼年细胞）起,与芹菜素（1、3、10μmol·L^-1）共孵育。通过检测细胞β-半乳糖苷酶（sAβ-gal）阳性率,推断细胞衰老程度;通过检测胞内双氢罗丹明氧化产物Rh123的荧光强度,推断胞内活性氧簇（ROS）水平,即氧化应激水平;通过检测培养液中硝酸盐／亚硝酸盐含量,推断胞内一氧化氮（NO）水平。结果：与幼年细胞组相比,老年细胞对照组中内皮细胞SAβ-gal阳性率及ROS水平均明显提高（P〈0．05）,且其培养液中硝酸盐／亚硝酸盐含量明显降低（P〈0．05）;在老年细胞组中,与对照组相比,芹菜素组中内皮细胞sAβ-gal阳性率及ROS水平均明显呈芹菜素剂量依赖性降低（P〈0．05）,而其培养液中硝酸盐／亚硝酸盐含量也明显呈芹菜素剂量依赖性增加（P〈0．05）。结论：芹菜素可延缓人脐静脉内皮细胞的衰老,且该作用与胞内ROS生成减少及NO生成增加有关。     

人脐静脉血管内皮细胞的高效分离与培养

目的:建立人脐静脉内皮细胞(HUVEC)体外分离、培养、鉴定的高效方法.方法:胰酶消化法从脐静脉获得HUVEC,观察其形态并传代培养,检测其摄取Dil标记的乙酰化低密度脂蛋白(Dil-AcLDL)的情况,传3代流式榆测内皮细胞特异性表达.结果:消化分离的细胞呈小圆形且聚集成团;2h细胞开始贴壁,呈条索状;7d细胞增多呈漩涡样生长;此后细胞继续增多接近融合,呈铺路石样改变.流式检测细胞高表达血管内皮细胞特异性标志CD144、vWF、CD31,部分表达CD34,而白细胞共同抗原CD45为阴性表达.镜下发现贴壁细胞原代、第5代摄取Dil-AcLDL,胞浆呈现红色荧光,提示为有活性的内皮细胞.结论:利用胰酶消化法能从人脐静脉获得大量内皮细胞,体外培养后能成功增殖传代.     

萝卜硫素通过Src/PI3K/Akt通路调控人脐静脉内皮细胞NO的生成机制

目的：研究萝卜硫素（SFN）促进人脐静脉内皮细胞（human umbilical vein endothelial cells,HUVEC）生成一氧化氮（NO）,参与内皮细胞修复的作用机制。方法：采用MTT法检测SFN对HUVEC细胞存活率的影响;检测SFN对HUVEC NO释放量的影响;采用Western blot法检测SFN对NO释放途径相关蛋白表达量的影响。结果：SFN浓度低于50 μmol·L^-1时,对HUVEC细胞无明显毒性（P<0.05）;SFN可显著增加NO释放量,呈剂量依赖性,eNOS特异性抑制剂L-NAME明显抑制NO生成（P<0.05）;SFN呈时间和剂量依赖性显著增加eNOS磷酸化（p-eNOS）水平,促使Akt磷酸化（p-Akt）及PI3K上游调节因子Src激酶的活化。结论：SFN通过Src/PI3K/Akt信号通路增强HUVEC细胞中eNOS的活性,促进NO生成参与内皮细胞修复,为SFN在预防内皮功能障碍、动脉粥样硬化和其他心血管疾病方面的应用提供实验依据。     

MK对人脐静脉内皮细胞增殖及迁移的影响

目的 通过研究midkine（MK）蛋白对人脐静脉内皮细胞（HUVEC）增殖、迁移的影响,阐明MK蛋白在血管生成中的作用.方法 选择HUVEC为研究对象进行细胞常规培养,分为5组分别给予不同浓度MK进行干预,实验组MK浓度分别为0.5、5、50和500 ng/ml,以未处理组作为对照组.利用CCK-8试剂盒检测MK对HUVEC增殖能力的影响,利用Transwell技术检测MK对HUVEC迁移能力的影响.结果 与对照组相比,5 ng/ml的MK作用24h和48 h可以促进细胞增殖,差异有统计学意义（P＜0.05）;5 ng/ml的MK作用24 h可以促进细胞迁移,差异有统计学意义（P＜0.05）.结论 一定浓度的MK作用于内皮细胞,可以促进其增殖和迁移,提示MK在血管生成中具有重要作用.     

sCD40L对人脐静脉内皮细胞分泌TNF-α的影响

目的：探讨sCD40L对人脐静脉内皮细胞（ HUVEC）分泌TNF-α的影响。方法分组培养HUVEC,应用不同浓度（0、10、20、30、40μg/L）、不同时间（0、10、20、30、40 min）人重组CD40L（ r-CD40L）刺激人脐静脉内皮细胞,采用酶联免疫吸附法（ ELLISA）测定细胞培养上清液中TNF-α的浓度。结果在相同浓度r-CD40L（20μg/L）刺激下,HUVEC在20 min分泌TNF-α水平最高,5组间比较差异有统计学意义（F ＝500．15, P ＜0．01）;随r-CD40L刺激浓度增加,HUVEC分泌TNF-α水平增高,呈浓度依赖性,5组间比较差异有统计学意义（ F ＝128．75, P ＜0．01）。结论 HUVEC可在r-CD40L刺激的早期分泌TNF-α达峰值,r-CD40L刺激HUVEC分泌TNF-α呈浓度依赖性。     

银杏黄酮苷对氧化损伤内皮细胞产生NO、eNOS和ICAM-1的影响

目的观察银杏黄酮苷对氧化损伤的人脐静脉内皮细胞（HUVEC）产生NO、内皮型一氧化氮合酶（eNOS）和人可溶性细胞间黏附分子（ICAM-1）的影响。方法体外培养HUVEC,传至3代后,以不同浓度的银杏黄酮苷分别作用于HUVEC,然后进行氧化损伤处理。以硝酸还原酶法测定培养液上清中的NO水平,免疫细胞化学法检测内皮细胞eNOS的表达,ELISA法测定细胞培养液中ICAM-1的含量。结果HUVEC在氧化损伤（H2O2100μmol/L,2h）后产生NO的量显著减少（P〈0.01）,eNOS表达下调,ICAM-1表达上调;银杏黄酮苷可以剂量依赖性的增加内皮细胞NO生成量,上调eNOS的表达,下调ICAM-1表达。结论银杏黄酮苷可能通过增加HUVEC eNOS的表达增加N0的释放、抑制ICAM-1的表达等机制对内皮细胞起保护作用。     

Effect of beta-escin sodium on endothelial cells proliferation, migration and apoptosis

beta-Escin, the major active compound in extracts of the horse chestnut Aesculus hippocastanum seed, has shown clinically significant activity in chronic venous insufficiency (CVI). Our previous studies had shown that beta-escin sodium inhibited angiogenesis in chick chorioallantoic membrane (CAM) and in aortic disk assay. In this study, we explored the direct effect of beta-escin sodium on proliferation, migration and apoptosis in human umbilical vein endothelial cells (HUVECs) and ECV304 cells. Sulforhodamine B (SRB) assay showed that beta-escin sodium (10, 20, 40 microg/ml) inhibited endothelial cells (ECs) proliferation dose-dependently. beta-escin sodium also induced ECs apoptosis at 40 microg/ml. Cell migration was evaluated by an improved wound assay: barren spot assay. And the direct effect on cell motility excluding influence of cell proliferation was examined by High Content Screening (HCS, Cellomics) assay. The data indicated that beta-escin sodium suppressed ECs migration and cell motility. Western blot results suggested that beta-escin sodium acts on ECs possibly by increasing expression of thrombospondin-1 (TSP-1), and decreasing expression of PKC-alpha and activation of p44/42 mitogen-activated protein kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAPK). Our findings give the evidence that beta-escin sodium might have potential anti-angiogenic activity via its direct effects on ECs.     

Effect of β-escin sodium on endothelial cells proliferation, migration and apoptosis

β-Escin, the major active compound in extracts of the horse chestnut Aesculus hippocastanum seed, has shown clinically significant activity in chronic venous insufficiency (CVI). Our previous studies had shown that β-escin sodium inhibited angiogenesis in chick chorioallantoic membrane (CAM) and in aortic disk assay. In this study, we explored the direct effect of β-escin sodium on proliferation, migration and apoptosis in human umbilical vein endothelial cells (HUVECs) and ECV304 cells. Sulforhodamine B (SRB) assay showed that β-escin sodium (10, 20, 40 μg/ml) inhibited endothelial cells (ECs) proliferation dose-dependently. β-escin sodium also induced ECs apoptosis at 40 μg/ml. Cell migration was evaluated by an improved wound assay: barren spot assay. And the direct effect on cell motility excluding influence of cell proliferation was examined by High Content Screening (HCS, Cellomics) assay. The data indicated that β-escin sodium suppressed ECs migration and cell motility. Western blot results suggested that β-escin sodium acts on ECs possibly by increasing expression of thrombospondin-1 (TSP-1), and decreasing expression of PKC-α and activation of p44/42 mitogen-activated protein kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAPK). Our findings give the evidence that β-escin sodium might have potential anti-angiogenic activity via its direct effects on ECs.     

Study on the mechanisms of an extract of Salvia miltiorrhiza on the regulation of permeability of endothelial cells exposed to tumour necrosis factor-alpha

Exposure of endothelial cells to tumour necrosis factor-alpha (TNF-alpha) results in increased endothelial permeability, accompanied by a loss of cell-cell adherence junctions. The importance of tyrosine phosphatase and kinase activity in oxidant-mediated loss of cell junction structures has been demonstrated. The purpose of this study was to determine whether tyrosine phosphorylation contributes to TNF-alpha-mediated disorganization of endothelial cell junctions and how an extract of Salvia miltiorrhiza (ESM) and its active ingredients, Danshensu (DSS) and salvianolic acid B (Sal B), exert their protective effect in maintaining cell integrity. Immunoblotting results indicated that TNF-alpha exposure resulted in tyrosine phosphorylation of junctional proteins such as vascular endothelial cadherin and beta-catenin, which was attenuated by ESM and its active ingredients DSS and Sal B. In addition, immunoprecipitation showed ESM and its active ingredients prevented beta-catenin disassociation from the cytoskeleton in TNF-alpha-treated human umbilical vein endothelial cells. The results suggest that TNF-alpha produced biological effects at least partly by junctional protein phosphotyrosine modifications by increasing the total cellular phosphorylation level. It could be concluded that ESM and its active ingredients were effective at eliminating the factors leading to the rise in cellular phosphorylation, thus helping to maintain the integrity of endothelial junction structure.     

蕲蛇酶对血管内皮细胞纤维蛋白溶解功能的影响

目的　研究蕲蛇酶对体外培养人脐静脉内皮细胞纤溶活性的影响 ,探讨其抗栓作用机制。方法　采用人脐带来源的脐静脉内皮细胞进行原代及传代培养 ,以光镜、电镜、第Ⅷ因子相关抗原免疫组化等方法鉴定内皮细胞 ,采用发色底物法 (S2 2 51 )测定内皮细胞培养液中组织型纤溶酶原激活剂(t PA)及组织型纤溶酶原激活剂抑制物 (PAI)的活性 ,ELISA法测定培养液中纤维蛋白降解产物的含量。结果　所培养的细胞具有特征性Weibel palade小体 ,第Ⅷ因子特异性抗原阳性。蕲蛇酶使细胞培养液中t PA活性升高 ,t PA/PAI比值升高 ,纤维蛋白降解产物明显增加。结论　蕲蛇酶具有促进血管内皮细胞释放t PA ,提高其纤溶活性的作用。     

MG132诱导人血管内皮细胞凋亡及对caspase-3表达的影响

目的　观察蛋白酶体抑制剂MG132对人血管脐静脉内皮细胞(ECV -304)的致凋亡作用及其对凋亡相关的天冬氨酸特异的半胱氨酸蛋白酶3表达的影响。方法　采用两个浓度(2, 5μmol·L-1 )的蛋白酶体抑制剂MG132处理ECV -304细胞;DNA琼脂糖凝胶电泳检测细胞凋亡,流式细胞术检测细胞周期和细胞凋亡率;RT -PCR检测细胞内凋亡相关基因caspase -3的转录水平;免疫细胞化学检测细胞caspase 3蛋白表达。结果　对照组ECV -304细胞凋亡率低于5%,在2μmol·L-1MG132作用下,凋亡率为11. 3%,MG132浓度升至5μmol·L-1,细胞凋亡率增致44 .5%,MG132诱导ECV 304细胞凋亡具有量-效关系;RT -PCR检测发现细胞内凋亡相关基因caspase 3mRNA表达上调;免疫细胞化学检测细胞caspase- 3蛋白表达水平升高。结论:蛋白酶体抑制剂MG132能够诱导血管内皮细胞凋亡,其机制可能与MG132抑制UPP活性,促进caspase- 3基因转录,使细胞内caspase -3增加而促进细胞凋亡。     

Atypical cannabinoid stimulates endothelial cell migration via a Gi/Go-coupled receptor distinct from CB1, CB2 or EDG-1

The endothelium-dependent mesenteric vasorelaxant effect of anandamide has been attributed to stimulation of a Gi/Go-coupled receptor, for which the nonpsychoactive analog abnormal cannabidiol (abn-cbd, (-)-4-(3-3,4-trans-p-menthadien-[1,8]-yl)olivetol) is a selective agonist and the compound O-1918 ((-)-4-(3-3,4-trans-p-menthadien-(1,8)-yl)-orcinol) is a selective antagonist. In human umbilical vein endothelial cells abn-cbd was reported to increase the phosphorylation of p44/42 mitogen activated protein kinase (MAPK) and protein kinase B/Akt, and these effects could be inhibited by pertussis toxin, by phosphatidylinositol 3-kinase (PI3K) inhibitors or by O-1918 [Mol. Pharmacol. 63 (2003) 699]. In the present experiments, abn-cbd caused a concentration-dependent increase in human umbilical vein endothelial cell migration, as quantified in a transwell chamber. This effect was antagonized by O-1918, by the PI3K inhibitor wortmannin, and by pertussis toxin, but not by the cannabinoid CB1 receptor antagonist AM251 or the cannabinoid CB2 receptor antagonist SR144528. The EDG-1 receptor agonist sphingosine-1-phosphate also increased human umbilical vein endothelial cell migration, but this response was unaffected by O-1918. In Chinese hamster ovary cells stably transfected with the gene encoding the EDG-1 receptor, p44/42 MAPK phosphorylation was unaffected by abn-cbd, but strongly induced by sphingosine-1-phosphate. These results indicate that an abn-cbd-sensitive endothelial receptor distinct from cannabinoid CB1, CB2 or EDG-1 receptors mediates not only vasorelaxation but also endothelial cell migration.     

HPLC-MS/MS 测定大鼠肝脏组织中丹参素钠的浓度

目的 建立液质联用色谱法(HPLC-MS/MS)测定SD大鼠肝脏组织中丹参素钠的浓度。 方法 肝脏组织样品制成匀浆后,加入内标酮洛芬,采用液液萃取方法进行预处理。色谱柱:Diamonsil C₁₈柱(200 mm×4.6 mm ,5 μm);流动相:甲醇-水(含0.01‰甲酸)(80:20);流速:1.0 ml/min;质谱条件:采用ESI离子源,负离子检测,选择MRM 测定丹参素钠和酮洛芬197→135 m/z 和 253→209 m/z。 结果 丹参素钠在20.2~1 010 n     

Attaching the phage display-selected GLA peptide to liposomes: factors influencing target binding

In our previous study, phage display selections were performed by in situ perfusion of a random peptide library through a mouse brain. This yielded two peptides (GLA and GYR) that showed significant binding to human brain endothelial cells (hCMEC/D3) when displayed on phage particles, but not to human umbilical vein endothelial cells (HUVECs). In the present study, these peptides were produced synthetically and coupled to liposomes to investigate the capacity of the peptides to act as ligands for targeting to hCMEC/D3 cells. Flow cytometry studies showed that these peptides when coupled to liposomes showed weak binding to the target brain endothelial cells. We hypothesized that the weak endothelial cell binding of the selected peptides when coupled to liposomes as compared to the binding of the peptides displayed on phage particles may be ascribed to: change of vehicle shape, change of peptide density, or change of peptide conformation. Peptide density on the liposomes influenced binding of the liposomes to the cells, however, this effect was minor. To study the influence of the peptide conformation, the GLA peptide was recombinantly produced fused to the N1-N2 domains of the phage p3 minor coat protein (p3-GLA) to mimic its conformation when displayed on phage particles. Binding of liposomes modified with either the GLA peptide or the p3-GLA protein to hCMEC/D3 cells was studied, and the p3-GLA-liposomes showed a higher binding to the cells compared to the GLA-liposomes. The experiments demonstrate that bringing the GLA peptide into the original phage protein environment restores and improves the peptide binding capacity and suggest that the GLA peptide, with some modifications, may be used as a brain-targeting ligand in the future.     

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。