蝎毒多肽提取物的抗血管生成作用

用不同浓度的东亚钳蝎素的多肽提取物PESV(4～20μg／ml)作用于人脐静脉内皮细胞(HUVEC),观察HUVEC增殖活性和凋亡变化,增殖活性检测采用BrdU掺入的ELISA法,凋亡水平和凋亡相关基因Bcl-2和Bax表达的检测采用流式细胞术检测;用鸡胚尿囊膜(CAM)显示PESV对血管生成的抑制作用。结果显示,PESV抑制HUVEC的增殖,而对乳腺癌细胞MDA-MB-231的增殖无明显影响;PESV作用72h后,HUVEC凋亡相关基因Bcl-2表达降低,Bax表达增加,凋亡细胞比例增至10．5％,明显高于对照组;0．5mgPESV能明显抑制CAM新生血管的形成。因此,PESV具有良好的体外抗肿瘤血管生成活性,PESV作为一种肿瘤血管抑制剂的天然药物来源,其有效成分和药理作用有待进一步研究。     

肿瘤血管生成抑制剂的作用机制研究进展

肿瘤血管生成抑制剂批能破坏或抑制血管生成,有效地阻止肿瘤生长和转移的药物,可分为特异性和非特异性两大类。其作用机制主要有：（１）调控血管形成生长因子;（２）抑制基底膜降解;（３）影响信号转导通路;（４）调控细胞生长周期;（５）调控肿瘤机关基因。本文对其作用机制的进展作一综述。     

阿魏酸在缺氧条件下促进人脐静脉内皮细胞血管形成的实验研究

目的:研究阿魏酸(ferulic acid,FA)在缺氧条件下对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)增殖、迁移和管腔样结构形成的影响。方法:原代培养人脐静脉内皮细胞,在缺氧实验条件下,细胞被分为7组,即1个对照组和6个实验组。对照组采用1%酒精处理,实验组用不同浓度(1×10~(-8)、1×10~(-7)、1×10~(-6)、1×10~(-5)、1×10~(-4)及1×10~(-3) mol/L)的阿魏酸处理。分别采用MTS法、划痕法、Matrigel法分析不同浓度阿魏酸处理对人脐静脉内皮细胞的增殖、迁移和管腔样结构形成的影响。结果:缺氧条件下,浓度为1×10~(-6)~1×10~(-4)mol/L的阿魏酸处理能明显促进HUVECs的增殖(P0.05),以1×10~(-5) mol/L处理的效果最好(P0.01);与对照组相比,1×10~(-6)mol/L(P0.05)、1×10~(-5) mol/L(P0.01)及1×10~(-4) mol/L(P0.01)阿魏酸处理均能明显促进HUVECs横向迁移,以1×10~(-5) mol/L处理迁移的细胞数量最多;1×10~(-8)~1×10~(-4) mol/L阿魏酸处理能不同程度地促进HUVECs管腔样结构的形成,以1×10~(-5) mol/L处理形成管腔样结构的数量最多(P0.01)。结论:阿魏酸在缺氧条件下能促进人脐静脉内皮细胞的增殖、迁移和管腔样结构形成。     

干细胞外泌体对内皮细胞增殖迁移和血管生成的影响

[目的]探究脂肪间充质干细胞来源的外泌体对人脐静脉内皮细胞的迁移和血管生成的影响。[方法]利用超速离心法提取脂肪间充质干细胞来源的外泌体;通过透射电子显微镜观察外泌体的形貌;通过免疫印迹法检测外泌体的标志蛋白;通过动态光散射法检测外泌体水合粒径;通过CCK-8法检测外泌体对HUVECs增殖的作用;通过划痕实验和Transwell实验检测外泌体对HUVECs迁移的影响;通过血管生成实验评估外泌体诱导HUVECs生成血管的能力。[结果]脂肪间充质干细胞外泌体的平均水合粒径约为151.9±12.3 nm,含有外泌体的标志蛋白CD63、TSG101。外泌体浓度为30μg/mL时,共孵育48 h后,EXO组HUVECs的增殖率高于NC组14%;划痕实验中NC组的平均迁移率约为0.45±0.05,EXO组约为0.63±0.05,EXO组的迁移率显著高于NC组约为0.18±0.07,而Transwel中48 h时NC组的单位面积平均转移细胞数约为167±24,EXO组约为728±49。4 h的成血管实验显示外泌体组管的NC组平均结点数约为495±52,EXO组约为658±76;NC组单位图像平均分支...     

衰老对AhR 及其血管生成相关因子表达的影响

目的:探讨芳香烃受体(aryl hydrocarbon receptor,Ah R)在过氧化氢(H_2O_2)诱导的人脐静脉内皮细胞(HUVECs)衰老模型中表达的变化情况及其对血管生成相关因子表达的影响。方法:采用不同浓度的H_2O_2(100μM,200μM,300μM)处理HUVECs诱导内皮细胞衰老,通过β-半乳糖苷酶染色观察细胞衰老情况,Western blot检测HUVECs中Ah R蛋白以及血管生成相关因子低氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)蛋白表达,ELISA检测细胞培养上清液中血管内皮生长因子(vascular endothelial growth factor,VEGF)表达的变化。结果:与对照组相比,经过不同浓度H_2O_2处理后,HUVECs均出现细胞衰老表现,Ah R蛋白表达显著增加,HIF-1α以及VEGF蛋白表达明显减少,且均呈现出剂量依赖性,以300μM H_2O_2处理组最为显著。结论:在过氧化氢诱导衰老的人脐静脉内皮细胞中Ah R蛋白表达明显增加,HIF-1α和VEGF等促血管生成因子明显减少。     

丹红注射液中5种主要活性成分促血管新生作用研究

目的:研究丹红注射液(DHI)入血后5个主要成分的促血管新生活性。方法:采用人脐静脉内皮细胞(HUVEC)正常培养体系和鸡胚绒毛尿囊膜(CAM)模型评价。细胞和鸡胚分别随机分为正常对照组,DHI组、羟基红花黄色素A组、丹酚酸B组、丹参素组,原儿茶醛组和迷迭香酸组。MTT法检测HUVEC增殖情况,免疫印迹法测定HUVEC促血管新生相关因子的表达,显微镜下计数鸡胚CAM给药区域新生血管数。结果:与正常对照组比较,10%(V/V)DHI及相应浓度的各单体均能显著增加HUVEC的增殖(P0.01),并促进HUVEC中促血管新生因子VEGF-A,b FGF,HGF的表达(P0.05,P0.01);而50%(V/V)DHI及相应浓度的各单体均能不同程度地增加鸡胚CAM给药区域新生血管数;两个筛选模型中,羟基红花黄色素A和丹参素均呈现一定的作用优势。结论:DHI入血后的5个主要活性成分都有促血管新生作用,其中羟基红花黄色素A和丹参素活性更佳。     

丹参酮ⅡA对正常和氧化损伤内皮细胞的保护作用研究

采用过氧化氢诱导建立的内皮细胞氧化损伤模型,探讨了丹参酮ⅡA对氧化损伤的HUVECs的保护作用.通过内皮细胞LDH、SOD、NOS、GSH-Px的活性和MDA、NO含量的变化趋势,MTT和流式细胞术反映的细胞存活率的变化,研究了丹参酮ⅡA对正常和氧化损伤HUVECs的作用.结果表明丹参酮ⅡA在10～100μmol/L浓度之间时,无明显细胞毒性.浓度在20 μmol/L以上时,丹参酮ⅡA能显著增强HUVECs抵御氧化损伤的能力;同时降低了LDH的泄漏,阻止了MDA等过氧化物的生成,提高了NOS、NO、SOD和GSH-PX分泌量,显著减少了细胞早期凋亡率和坏死率.表明丹参酮ⅡA可以保护HUVECs免受氧化损伤,防止动脉粥样硬化斑块的形成.     

脂联素抑制t-BHP诱导的内皮细胞凋亡作用及其可能机制探讨

目的：探讨重组脂联素对第三丁基过氧化氢（t-BHP）诱导的人脐静脉内皮细胞（HUVECs）凋亡的影响及其相关的分子机制。方法：以HUVECs作为研究对象,给予t-BHP处理,模拟体外HUVECs氧化损伤细胞凋亡模型。在此基础上,用携带重组脂联素基因的腺病毒转染HUVECs,观察重组脂联素对t-BHP诱导的HUVECs凋亡的影响。用MTT法检测细胞增殖活力。Hochest/PI荧光染色检测细胞凋亡率。Western blot检测细胞凋亡相关蛋白p-JNK、JNK和Caspase 3表达水平的变化。结果：100 μmol/L的t-BHP作用8 h可诱导HUVECs发生凋亡。与对照组相比,t-BHP组p-JNK、active caspase 3表达增多（P<0.01）。HUVECs高表达重组脂联素基因后,可明显抑制t-BHP诱导的HUVECs凋亡（P<0.01）,下调t-BHP诱导的p-JNK、active caspase 3表达。结论：持续t-BHP氧化损伤可诱导HUVECs发生凋亡。重组脂联素可有效抑制t-BHP诱导的HUVECs凋亡,其机制与p-JNK、active caspase 3的表达下调有关。     

番茄红素对血管内皮细胞氧化应激损伤的作用及机制研究

目的:观察番茄红素(lycopene,LYC)对于血管内皮细胞功能的作用,探讨其作用机制。方法:人脐静脉内皮细胞(HUVECs)处理实验分组:对照组,H2O2组,H2O2+LYC组(1、2、4、8μmolL-1)。MTT法检测HUVECs存活率;免疫印迹法(Western blot)检测p38MAPK蛋白磷酸化水平、抗凋亡蛋白B淋巴细胞/白血病-2(bcl-2)及线粒体凋亡通路相关蛋白bax的表达;细胞黏附能力测定和伤口愈合实验检测HUVECs粘附率和迁移率;TUNEL法检测HUVECs凋亡率;ELASA法测定HUVECs内活性氧(ROS),超氧化物歧化酶(SOD),乳酸盐脱氢酶(LDH)释放量和caspase-3的活性。结果:H2O2损伤后HUVECs存活率显著降低(P0.01),凋亡率显著增加(P0.01),黏附和迁移能力显著降低(P0.01),bax和p-p38MAPK的表达上调,bcl-2的表达下调,并且ROS、LDH的释放和caspase-3的活性增加(P0.01),SOD的释放减少。而LYC的预处理可以明显逆转H2O2以上作用。结论:H2O2氧化应激损伤中,LYC保护内皮细胞可能与其抗过氧化损伤细胞凋亡,抑制异常的p38MAPK信号通路有关。     

血管生长素促血管生成机制及其与疾病关系的研究进展

血管生长素(angiogenin,ANG)是一种促使新血管生成的诱导剂,它属于核糖核酸酶超家族中的一员。ANG蛋白含123个氨基酸,相对分子量为14．4kD,在肿瘤细胞中首先发现,也存在于正常细胞中,可由多种细胞分泌,其核糖核酸酶活性较弱。研究证实,ANG可以在细胞间及细胞内发挥促血管生长作用。ANG与缺血性疾病、肿瘤、及其它疾病的关系已见不少报道。本文简述ANG促血管生成作用机制及其与疾病的关系。     

血管生成抑制因子SCAIF80及其抗肿瘤效应

从鲨鱼软骨中提取、纯化出一种新的血管生成抑制因子－－鲨鱼软骨血管生成抑制因子80（shark carti-lage-derived angiogenesis inhibitory factor 80,SCAIF80）,SDS－PAGE银染分析显示一条带,分子量为80kD。利用血管内上细胞增殖、迁移抑制实验,证明SCAIF80能显著抑制血管内皮细胞的增殖与迁移,均有剂量依赖性。采用鸡胚绒毛尿囊膜分析,证明SCAIF80能显著抑制新生血管生成,动物抑瘤实验结果表明SCAIF80显著抑制小鼠肿瘤生长,对Lewis肺癌的抑瘤率达93.83%。上结果提示鲨鱼软骨含有一种新的蛋白质,可抑制新生血管生成,并因此抑制肿瘤生长。     

腺苷对人脐静脉内皮细胞三维管状结构形成的影响

目的:探讨腺苷对脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)三维管状结构形成的影响.方法:建立上、下两层内皮细胞,中间为胶原凝胶的三维培养方式.设对照和试验各3孔.对照孔不加腺苷,实验孔内加入10-4mol/L腺苷.观察并记录特定视野下芽生的管状结构数目.结果:HUVEC可以在Ⅰ型胶原凝胶中形成三维网状结构,腺苷实验组细胞生长快,出芽快,管状结构粗大,甚可形成贯穿胶原的三维网状结构.血管芽生数与对照组比较在24 h、48 h、72 h、96 h均有统计学差异(P＜0.05或P＜0.01).结论:腺苷对HUVEC三维网状结构的形成有促进作用.     

TWIST1在人脐静脉内皮细胞增殖、迁移及血管形成中的作用

探讨TWIST1在原代人脐静脉内皮细胞（human umbilical vein endothelial cells,HUVECs）增殖、迁移及体外血管生成中的作用。用有靶向人TWIST1基因shRNA（pLL3.7-shTwist1-GFP）的慢病毒液感染试验组细胞,同时以携带Scramble shRNA的慢病毒液（pLL3.7-shCtrl-GFP）感染对照组细胞,用流式细胞术测定细胞感染效率,实时荧光定量PCR（real-time fluorescent quantitative PCR,qRT-PCR）检测shRNA的基因沉默效率。通过制作细胞生长曲线、Annexin V/7AAD染色流式细胞术、细胞划痕实验、小管形成实验、qRT-PCR检测TWIST1表达降低对HUVECs的增殖、凋亡、迁移、血管形成能力以及血管生长因子受体2（vascular endothelial growth factor receptor 2,VEGFR2）基因表达的影响。试验组TWIST1基因表达下降为对照组的30%,表明shTWIST1能有效降低TWIST1基因的表达。与对照组相比,敲降TWIST1能明显抑制HUVECs的增殖（P<0.01）,诱导细胞凋亡（P<0.05）。试验组HUVECs划痕愈合率、体外生成的血管样结构数目和总小管分支长度均显著低于对照组（P<0.01）;与对照组相比,试验组HUVECs中VEGFR2的表达显著降低（P<0.01）。通过探究HUVECs表达的TWIST1在内皮细胞增殖、存活、迁移和毛细血管样结构的形成中的作用,为TWIST1作为抑制肿瘤血管新生治疗的新靶点提供一定的理论依据。     

Synergistic effect of extracellularly supplemented osteopontin and osteocalcin on stem cell proliferation,osteogenic differentiation,and angiogenic properties

A high demand for functional bone grafts is being observed worldwide, especially due to the increased life expectancy. Osteoinductive components should be incorporated into functional bone grafts, accelerating cell recruitment, cell proliferation, angiogenesis, and new bone formation at a defect site. Noncollagenous bone matrix proteins, especially osteopontin (OPN) and osteocalcin (OC), have been reported to regulate some physiological process, such as cell migration and bone mineralization. However, the effects of OPN and OC on cell proliferation, osteogenic differentiation, mineralization, and angiogenesis are still undefined. Therefore, we assessed the exogenous effect of OPN and OC supplementation on human bone marrow mesenchymal stem/stromal cells (hBM MSC) proliferation and osteogenic differentiation. OPN dose-dependently increased the proliferation of hBM MSC, as well as improved the angiogenic properties of human umbilical vein endothelial cells (HUVEC) by increasing the capillary-like tube formation in vitro. On the other hand, OC enhanced the differentiation of hBM MSC into osteoblasts and demonstrated an increase in extracellular calcium levels and alkaline phosphatase activity, as well as higher messenger RNA levels of mature osteogenic markers osteopontin and osteocalcin. In vivo assessment of OC/OPN-enhanced scaffolds in a critical-sized defect rabbit long-bone model revealed no infection, while new bone was being formed. Taken together, these results suggest that OC and OPN stimulate bone regeneration by inducing stem cell proliferation, osteogenesis and by enhancing angiogenic properties. The synergistic effect of OC and OPN observed in this study can be applied as an attractive strategy for bone regeneration therapeutics by targeting different vital cellular processes.     

~ffects of endothelial microvesicles induced by A23187 on H9c2 cardiomyocytes

Objective To investigate the effects of endothelial microvesicles （EMVs） induced by calcium ionophore A23187 on H9c2 cardiomyocytes. Methods Human umbilical vein endothelial cells （HUVECs） were treated with 10 μmol/L A23187 for 30 min. EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium. EMVs were characterized using 1 and 2 Ilm latex beads and anti- PE-CD144 antibody by flow cytometry. For functional research, EMVs at different concentrations were co- cultured with H9c2 cardiomyocytes for 6 h. Cell viability of H9c2 cells and the activity of LDH leaked from H9c2 cells were tested by colorimetry. Moreover, apoptosis of H9c2 cells was observed through Hoechst 33258 staining and tested by FITC-Annexin V/Pl double staining. Results EMVs were induced by A23187 on HUVECs, and isolated by ultracentrifugation. We identified the membrane vesicles （〈 1 μm） induced by A23187 were CD144 positive. In addition, the EMVs could significantly reduce the viability of H9c2 cells, and increase LDH leakage from H9c2 cells in a dose dependent manner （P〈0.05）. Condensed nuclei could be observed with the increasing concentrations of EMVs through Hoechst 33258 staining. Furthermore, increased apoptosis rates of H9c2 cells could be assessed through FITC-Annexin V/PI double staining by flow cytometry. Conclusion Microvesicles could be released from HUVECs after induced by A23187 through calcium influx, and these EMVs exerted a pro-apoptotic effect on H9c2 cells by induction of apoptosis.     

裂解型腺病毒Ad-E1A对人脐静脉内皮细胞的影响

以QBI-293A细胞基因组DNA为模板,PCR扩增E1A基因,酶切连接到pAdTrack-CMV转移质粒上,pAdTrack-CMV-E1A经PmeI线性化后,与pAdEasy-1共转化大肠杆菌BJ5183,筛选重组腺病毒质粒pAdEasy-1-pAdTrack-CMV-E1A,经PacI线性化,脂质体转染QBI-293A细胞,获得裂解型腺病毒Ad-E1A。裂解型腺病毒Ad-E1A在ECV304细胞内复制裂解,抑制细胞的生长,并可以降低VEGF的表达,探讨了Ad-E1A可能通过抑制ECV304细胞NF-κB的激活而引起细胞生长抑制的机制,说明Ad-E1A具有抑制肿瘤转移的功能。     

Characterization of a novel angiogenic model based on stable, fluorescently labelled endothelial cell lines amenable to scale-up for high content screening

Background. Blood vessel formation is important for many physiological and pathological processes and is therefore a critical target for drug development. Inhibiting angiogenesis to starve a tumour or promoting ‘normalization’ of tumour vasculature in order to facilitate delivery of anticancer drugs are both areas of active research. Recapitulation of vessel formation by human cells in vitro allows the investigation of cell—cell and cell—matrix interactions in a controlled environment and is therefore a crucial step in developing HCS (high content screening) and HTS (high throughput screening) assays to search for modulators of blood vessel formation. HUVECs (human umbilical‐vein endothelial cells) exemplify primary cells used in angiogenesis assays. However, primary cells have significant limitations that include phenotypic decay and/or senescence by six to eight passages in culture, making stable integration of fluorescent markers and large‐scale expansion for HTS problematic. To overcome these limitations for HTS, we developed a novel angiogenic model system that employs stable fluorescent endothelial cell lines based on immortalized HMECs (human microvascular endothelial cell). We then evaluated HMEC cultures, both alone and co‐cultured with an EMC (epicardial mesothelial cell) line that contributes vascular smooth muscle cells, to determine the suitability for HTS or HCS. Results. The endothelial and epicardial lines were engineered to express a panel of nuclear‐ and cytoplasm‐localized fluorescent proteins to be mixed and matched to suit particular experimental goals. HMECs retained their angiogenic potential and stably expressed fluorescent proteins for at least 13 passages after transduction. Within 8 h after plating on Matrigel, the cells migrated and coalesced into networks of vessel‐like structures. If co‐cultured with EMCs, the branches formed cylindrical‐shaped structures of HMECs surrounded by EMC derivatives reminiscent of vessels. Network formation measurements revealed responsiveness to media composition and control compounds. Conclusions. HMEC‐based lines retain most of the angiogenic features of primary endothelial cells and yet possess long‐term stability and ease of culture, making them intriguing candidates for large‐scale primary HCS and HTS (of ～10000–1000000 molecules). Furthermore, inclusion of EMCs demonstrates the feasibility of using epicardial‐derived cells, which normally contribute to smooth muscle, to model large vessel formation. In summary, the immortalized fluorescent HMEC and EMC lines and straightforward culture conditions will enable assay development for HCS of angiogenesis.     

红杉醇对高糖诱导的人脐静脉内皮细胞NOX4、eNOS表达的影响

目的：观察红杉醇（Scq）对高糖诱导的人脐静脉内皮细胞（HUVECs）损伤的保护作用及机制。方法：原代培养HUVECs,红杉醇（0．1,1,10μmol/L）预处理1h后,30mmol/L葡萄糖诱导内皮细胞损伤。5-溴脱氧尿嘧啶核苷（BrdU）掺入法检测细胞增殖,流式细胞术检测细胞周期,2’7’-二乙酰二氯荧光素（DCFH-DA）免疫荧光法检测细胞内活性氧簇（R0s）水平,比色法检测细胞-氧化氮（NO）、丙二醛（MDA）及过氧化氢（H202）水平,real-timePCR和Westernblot检测细胞内皮型一氧化氮合酶（eNos）及NADPH氧化酶4（NOX4）mRNA和蛋白表达。结果：Seq预处理1h后能明显减轻高糖诱导的血管内皮细胞损伤,促进细胞增殖,降低胞内NOX4的表达及ROS、MDA及H202水平,上调eNOS的表达及NO水平。结论：Seq对高糖诱导的内皮细胞损伤具有一定的保护作用,其机制可能与其抗氧化、上调eNOS的表达有关。     

Inhibition of exosomal miR‐24‐3p in diabetes restores angiogenesis and facilitates wound repair via targeting PIK3R3

Diabetic foot ulcer (DFU) is one of the common ailments of elderly people suffering from diabetes. Exosomes containing various active regulators have been found to play a significant role in apoptosis, cell proliferation and other biological processes. However, the effect and the underlying mechanism of action of diabetes patients derived from circulating exosomes (Dia‐Exos) on DFU remain unclear. Herein, we aim to explore the potential regulatory role of Dia‐Exos. First, we attempted to demonstrate the harmful effect of Dia‐Exos both in vivo and in vitro. miRNA‐24‐3p (miR‐24‐3p) was found enriched with Dia‐Exos. Hence, inhibition of this miRNA could partially reverse the negative effect of Dia‐Exos, thus, in ture, accelerates wound repair. Luciferase assay further verified the binding of miR‐24‐3p to the 3′‐UTR of phosphatidylinositol 3‐kinase regulatory subunit gamma (PIK3R3) mRNA and the PIK3R3 expression enhanced human umbilical vein endothelial cells functionality in vitro. Hence, the findings of this study reveal the regulatory role of Dia‐Exos in the process of wound healing and provide experimental evidence for the therapeutic effects of knocking down miR‐24‐3p in DFU treatment.     

MG132抑制肝癌细胞Bel-7404生长的机制研究

MG132(Z-Leu-leu-leu-CHO) 是一种蛋白酶体抑制剂,它可逆地抑制蛋白酶体活性,从而抑制泛素-蛋白酶体通路所介导的蛋白质降解,诱导细胞凋亡．实验研究MG132抑制肝癌细胞Bel-7404生长的机制．采用不同浓度梯度和时间梯度,通过荧光显微镜、透射电子显微镜、Hoechst33342染色、MTT检测、AnnexinⅤ/PI流式细胞术、Western blot分别检测MG132 对Bel-7404细胞的形态学变化、细胞内质网压力变化、自噬泡形成、凋亡小体形成、细胞存活率、细胞凋亡水平、凋亡及自噬信号途径中相关蛋白质表达的影响．结果显示,MG132能明显抑制Bel-7404细胞生长．通过增加内质网压力激活Caspase-12,也可通过线粒体途径调节Bcl-2/Bax水平,促进细胞色素c的释放,两者皆可激活下游效应Caspase-3,剪切PARP,诱导细胞凋亡．同时,MG132可诱导Beclin 1 和LC3B的上调,促使Bel-7404细胞发生自噬,可在透射电镜下观察到自噬泡形成．上述结果表明,MG132作用Bel-7404细胞涉及两类细胞程序性死亡途径：细胞凋亡和自噬．