重组人促红细胞生成素对人脐静脉内皮细胞CyclinD1表达的影响

目的探讨重组人促红细胞生成素(rHuEPO)对人脐静脉内皮细胞(HUVEC)细胞周期蛋白CyclinD1的影响。方法采用细胞培养及免疫组织化学方法检测不同剂量(10、20、40U/mL)rHuEPO作用下HUVEC中CyclinD1表达的变化。结果对组照及10、20、40U/mlrHuEPO实验组内皮细胞CyclinD1表达阳性百分率(%)分别为26±3、41±5、69±3、65±4,与对照组相比,rHuEPO作用后HUVEC中CyclinD1表达明显增强(P﹤0.05),且随rHuEPO剂量增加作用增强,呈剂量效应关系。结论rHuEPO能明显促进体外培养内皮细胞CyclinD1表达,提示rHuEPO具促内皮细胞增殖作用。     

重组人促红细胞生成素对体外培养内皮细胞增殖的影响

目的:研究重组人促红细胞生成素(rhEPO)对体外培养内皮细胞增殖的影响.方法:从脐静脉体外分离培养人脐静脉内皮细胞(HUVEC),采用形态学观察及Ⅷ因子相关抗原(Ⅷ-R Ag)检测进行内皮细胞鉴定,免疫组织化学方法检测rhEPO对HUVEC增殖细胞核抗原(PCNA)与细胞周期蛋白D1(Cyclin D1)的表达变化.结果:rhEPO实验组内皮细胞PCNA与Cyclin D1表达均明显高于对照组.当rhEPO剂量提高到20U/ml时其作用最强.结论:rhEPO对内皮细胞增殖有明显促进作用,这种促进作用町能是其影响血管生成的主要原因之一.     

人血管内皮生长因子基因的克隆、表达及生物活性分析

目的 克隆人血管内皮细胞生长因子165(VEGF165)基因，构建真核表达载体，观察其对脐静脉内皮细胞的增殖作用和血管新生的影响。方法 利用RT-PCR方法，从人扁桃体组织中扩增人VEGF165 cDNA完整编码区，并构建成pcDNA3．1( )／VEGF165(简称pcDNA／V)重组体；应用脂质体介导的基因转移技术将构建的真核表达载体pcDNA／V体外转染至人脐静脉内皮细胞(HUVEC)，MTT法检测其对内皮细胞增殖的影响。建立家兔下肢缺血模型，注射重组质粒pcDNA／V，pcDNA3．1( )空质粒作对照，选取不同时间点，行血管造影。结果 构建的真核表达载体pcDNA／V的酶切电泳分析和测序表明结果正确。pcDNA／V转染HUVEC能明显促进内皮细胞的分裂增殖。血管造影显示，术后基因治疗组远端动脉充盈早于对照组，新生血管数目也明显多于同时期对照组。结论 成功克隆了人VEGF165基因，构建了其真核表达载体。体内外生物学活性研究证实，重组质粒的表达产物具有刺激HUVEC增殖和促进缺血肢体侧枝循环建立的功能。     

miR-203真核表达载体的构建和重组慢病毒的制备及其对人脐静脉内皮细胞增殖和迁移的影响

目的 构建含有人源microRNA-203(miR-203)的慢病毒表达载体并制备重组慢病毒,感染人脐静脉内皮细胞系(HUVEC-12),观察其对细胞增殖和迁移的影响.方法 使用PCR方法克隆miR-203前体分子的DNA片段,构建并包装过表达miR-203的重组慢病毒.利用慢病毒感染系统建立稳定过表达miR-203的人脐静脉内皮细胞株(HUVEC-Lv-miR-203).实时荧光定量PCR(qRT-PCR)检测外源导入miR-203的表达情况.MTT法检测miR-203对HUVEC体外生长的影响,利用划痕恢复实验检测miR-203对HUVEC迁移能力的影响.结果 成功构建了过表达miR-203重组慢病毒表达系统.MTT法证实miR-203可在体外抑制HUVEC增殖,划痕恢复实验结果提示该细胞株的迁移能力受到显著抑制.结论 miR-203可以抑制体外培养的HUVEC增殖和迁移.     

几种外用中药成份对离体人脐静脉内皮细胞增殖的影响

目的:体外观察几种常用外用中药有关成分对血管内皮细胞增殖作用的影响,初步探讨其对创伤愈合的可能作用。方法:采用MTT比色法观察中药人参皂苷Rg1、Rh1、黄芪多糖、鹿茸多肽、川芎嗪、麝香酮、桂皮醛和乳香水提物对人脐静脉内皮细胞(HUVEC)增殖的影响。结果:9.75mg/L-2.5g/L的黄芪多糖对HUVEC未表现出增殖促进作用;1.94mg/L-0.5g/L的人参皂苷Rh1促进HUVEC的增殖(P＜0.05或P＜0.01),31mg/L的人参皂苷Rg1抑制细胞增殖(P＜0.05);1mg/L-0.5g/L的鹿茸多肽明显促进HUVEC的增殖,以10mg/L作用最明显(P＜0.01),桂皮醛2g/L时促进HUVEC的增殖(P＜0.05);1g/L的麝香酮明显抑制HUVEC的增殖(P＜0.01);0.5kg/L-2.5kg/L(生药)乳香水提物明显抑制HUVEC的增殖(P＜0.01),抑制增殖率为35.56%-55.56%。川芎嗪0.125g/L-0.5g/L明显抑制HUVEC的增殖(P＜0.01)。结论:益气温阳药物人参(Rh1)、鹿茸(鹿茸多肽)、肉桂(桂皮醛)促进HUVEC增殖;通络活血药物麝香(麝香酮)、乳香(乳香水提物)、川芎(川芎嗪)抑制HUVEC增殖。     

罗格列酮对高糖环境下人脐静脉内皮细胞作用的研究

目的研究罗格列酮对高糖环境下人脐静脉内皮细胞促血管生成功能的影响及其机制。方法通过MTT检测法、细胞划痕实验,探讨罗格列酮在高糖环境下对人脐静脉内皮细胞(HUVEC)的增殖与迁移能力的影响,并用ELISA方法检测经罗格列酮作用后高糖培养基上清中VEGF、SDF-1的含量,进行统计分析。结果经罗格列酮药物处理后的HUVEC增殖和迁移能力明显高于高糖组,且培养基中VEGF和SDF-1的含量也有显著升高。AKT阻断剂可以阻断罗格列酮对HUVEC增殖、迁移和分泌功能的促进作用。结论罗格列酮在高糖环境下可显著促进HUVEC的增殖、迁移和分泌功能,AKT信号通路在这一过程中发挥着重要的作用。     

葛根素通过ERK1/2信号通路促进内皮细胞增殖

目的:体外观察葛根素对人脐静脉内皮细胞(HUVEC)的促增殖作用,并对其机制进行初步探讨。方法:采用四甲基偶氮唑蓝(MTT)法检测不同浓度葛根素对HUVEC细胞增殖的影响;流式细胞术检测细胞凋亡;Western blot检测p-ERK1/2蛋白表达水平。结果:葛根素能剂量依赖性促进HUVEC增殖,在80μM效果最明显,且作用36h时,能显著减少早期凋亡,激活ERK1/2磷酸化。结论:葛根素素对人脐静脉内皮细胞有明显的促进增殖作用,其作用可能与ERK1/2的活化有关。     

重组人促红细胞生成素对人脐静脉内皮细胞PCNA表达的影响

目的研究重组人促红细胞生成素（rHuEPO）对人脐静脉内皮细胞（HUVEC）增殖的影响。方法从脐静脉体外分离培养HUVEC，采用细胞培养及免疫组织化学方法检测不同剂量rHuEP0（10U／ml、20U／ml、40U／m1）对HUVEC增殖细胞核抗原（PCNA）表达的影响。结果对组照及10U／ml、20U／ml、40U／mlrHuEP0实验组内皮细胞PCNA阳性表达百分率分别为（31±3）％、（40±5）％、（68±6）％和（65±5）％，与对照组相比，rHuEPO作用后内皮细胞PCNA表达明显增强（P〈0．05），且随rHuEPO剂量提高作用增强，呈剂量效应关系。结论rHuEP0可促进体外培养内皮细胞增殖。     

津力达颗粒预处理对高糖诱导小鼠胰岛微血管内皮细胞和人脐静脉内皮细胞增殖及凋亡的影响

目的:观察中药津力达颗粒预处理对高糖诱导的小鼠胰岛微血管内皮细胞(MS-1)及人脐静脉内皮细胞(HUVEC)增殖及凋亡的影响。方法:体外培养MS-1及HUVEC株,均随机分为正常浓度葡萄糖(5.5mmol/L)培养组(正常对照组)、高浓度葡萄糖(33mmol/L)培养组(高糖组)、不同浓度津力达颗粒(12.5μg/ml、25μg/ml、50μg/ml、100μg/ml、200μg/ml、400μg/ml、800μg/ml)预处理24h后再高糖培养组(津力达组),各组平行培养48h后收集细胞,MTT法检测细胞增殖水平,流式细胞术检测细胞凋亡,包括总凋亡指数(TAI)和早期凋亡指数(EAI)。结果:与正常对照组比较,高糖组MS-1及HUVEC增殖水平(OD值)下降(P0.01),TAI和EAI升高(P0.01)。与高糖组比较,津力达组MS-1及HUVEC增殖水平随药物浓度增加而升高(P0.01),至津力达浓度800μg/ml时与正常对照组差异无统计学意义(P0.05)。津力达组MS-1和HUVEC的TAI及EAI则随药物浓度增加而降低(P0.01),但尚不能回复至正常对照组水平。结论:中药津力达颗粒升高MS-1及HUVEC增殖水平,降低MS-1及HUVEC的TAI和EAI,从而可能对胰岛功能及血管内皮功能具有一定保护作用。     

外用中药有效成分对血管内皮细胞增殖作用的影响

目的 :观察常用外用中药有效成分对血管内皮细胞增殖作用的影响 ,初步探讨其促进皮肤创伤愈合的机制。方法 :采用MTT比色法观察中药人参皂苷Rg1、Rh1、黄芪多糖、鹿茸多肽、麝香酮和桂皮醛、乳香水提物对人脐静脉内皮细胞 (HUVEC ,从健康产妇脐带中分离 )增殖的影响。结果 :在 2 4 4mg/L - 0 5g/L浓度范围内黄芪多糖对HUVEC未表现出增殖促进作用 ;在1 94mg/L - 0 5g/L浓度范围内人参皂苷Rh1促进HUVEC的增殖 (P <0 0 5或P <0 0 1) ,与对照组相比增殖率为 33 33% -111 11% ;在 1 94mg/L - 0 5g/L浓度范围内人参皂苷Rg1都有…     

Investigation of Interleukin 2 Receptors on Human Endothelial Cells

Abstract

We have demonstrated that both recombinant and purified IL-2 exert a direct effect on quiescent human microvascular endothelial cells in vitro, causing the cells to enter the cell cycle and proliferate (Hicks et al., 1989). In this study we have identified IL-2 receptors (R) on both human umbilical vein (HUVEC) and neonatal foreskin (HCEC) endothelial cells. The techniques used to identify the receptors included proliferation studies, flow cytometry and immunofluorescence. Results indicate that both HUVEC and HCEC possess low numbers of receptors since both cell types proliferate in response to IL-2. The number of receptors on the cell surface vary according to passage number and culture conditions. Immunofluorescent studies show discrete areas of staining on the cell membrane. These combined results suggest that human vascular endothelial cells possess IL-2R.     

Vascular endothelial cells provide T cells with costimulatory signals via the OX40/gp34 system

We investigated whether gp34, the ligand of OX40, expressed on EC is involved in costimulation of T cells. Normal CD4+ T cells were stimulated with anti-CD3-coated beads, phytohemagglutinin (PHA), or concanavalin A (Con A) in the presence or absence of irradiated human umbilical vein endothelial cells (HUVEC). Stimulation of T cells with each of these mitogens results in significant T-cell proliferation only when HUVEC were present, and this proliferation was inhibited markedly by anti-OX40 or anti-gp34 monoclonal antibody (mAb). T cells cultured with HUVEC produced more interleukin (IL)-2 than those cultured without HUVEC. The addition of anti-IL-2R alpha chain and anti-IL-2R beta chain mAbs abolished the costimulatory effects of HUVEC. Thus, the augmentation of T-cell proliferation appears to be attributable to increased IL-2 production. These results suggest that gp34 expressed on HUVEC plays a role in potentiation of T-cell immune response by providing OX40+ T cells with costimulatory signals.     

Human parvovirus B19 experimental infection in human fibroblasts and endothelial cells cultures

With the aim to detect what kind of cells, in addition to erythroid progenitors, could be involved in the pathogenesis of B19 infection in some connective tissue diseases, primary cultures of human fibroblasts (HF) and endothelial cells (HUVEC) were exposed to a B19 positive serum (350 genome copies/cell). The presence of NS1 and VP1 mRNA, in both HF and HUVEC cultures 1, 2 and 6 days after the exposure, indicated infection by B19 virus. However, no significant increase of B19 DNA level in the infected HF and HUVEC cultures was detectable through the entire incubation period of 6 days. It is possible that HF and HUVEC are not permissive for B19 virus replication or, alternatively, that few cells only get infected by B19 virus. HF and HUVEC stimulation with different growth factors or cytokines could be required for a B19 productive infection to occur.     

不同浓度哇巴因对人血管内皮细胞的作用

目的：研究不同浓度哇巴因对血管内皮细胞(HUVEC)的作用及生理浓度哇巴因激活的血管内皮细胞早期反应基因。方法： MTT法观察哇巴因对血管内皮细胞增殖的影响，台盼蓝染色及乳酸脱氢酶活性测定细胞活力。免疫组织化学染色法测定细胞核增殖抗原（PCNA）表达情况。以包含8 464条人类基因的DNA芯片检测血管内皮细胞受生理浓度哇巴因活化后的基因表达谱，并从中筛选出早期反应基因。 结果： 生理浓度（0.3-0.9 nmol/L）的哇巴因能刺激细胞增殖，且这种增殖不与剂量呈正相关，刺激增殖的最适浓度为0.3 nmol/L，最大效应作用时间为1-2 h。0.9-1.8 nmol/L的哇巴因抑制内皮细胞的增殖，引起细胞水肿和凋亡。但10 nmol/L的高浓度哇巴因却能够引起细胞的增殖。血管内皮细胞受哇巴因作用2 h后，基因表达谱研究显示340条基因出现表达差异，其中上调的共有145 条，多数与细胞代谢和转录调控相关，显著上调的6条。 结论： 哇巴因在参与维持血管内皮细胞的正常增殖及高血压引起的血管重塑中起重要作用。     

IKCa1通道阻滞剂TRAM-34对HUVEC增殖作用的影响

目的 体外应用IKCa1通道阻滞剂TRAM-34干预人脐静脉内皮细胞(HUVEC),探讨其抑制HUVEC增殖作用的影响.方法 采用免疫荧光和RT-PCR法观察HUVEC细胞IKCa1的表达,常规MTT法分析不同浓度TRAM-34作用24、48、72 h后HUVEC细胞增殖的变化.结果 免疫荧光和RT-PCR法均证实HU...     

The effect of human osteoblasts on proliferation and neo-vessel formation of human umbilical vein endothelial cells in a long-term 3D co-culture on polyurethane scaffolds

Angiogenesis is a key element in early wound healing and is considered important for tissue regeneration and for directing inflammatory cells to the wound site. The improvement of vascularization by implementation of endothelial cells or angiogenic growth factors may represent a key solution for engineering bone constructs of large size. In this study, we describe a long-term culture environment that supports the survival, proliferation, and in vitro vasculogenesis of human umbilical vein endothelial cells (HUVEC). This condition can be achieved in a co-culture model of HUVEC and primary human osteoblasts (hOB) employing polyurethane scaffolds and platelet-rich plasma in a static microenvironment. We clearly show that hOB support cell proliferation and spontaneous formation of multiple tube-like structures by HUVEC that were positive for the endothelial cell markers CD31 and vWF. In contrast, in a monoculture, most HUVEC neither proliferated nor formed any apparent vessel-like structures. Immunohistochemistry and quantitative PCR analyses of gene expression revealed that cell differentiation of hOB and HUVEC was stable in long-term co-culture. The three-dimensional, FCS-free co-culture system could provide a new basis for the development of complex tissue engineered constructs with a high regeneration and vascularization capacity.     

稳定表达组织型纤溶酶原激活因子基因的人脐静脉内皮细胞株的建立

为建立稳定表达组织型纤溶酶原激活因子(t-PA)基因的人脐静脉内皮细胞株,为其在基因修饰的组织工程血管中的应用奠定基础。首先构建t-PA基因的真核表达载体pcDNA3.1-Myc-HisB(-)/t-PA,将该表达载体用阳离子脂质体介导,转染人脐静脉内皮细胞系细胞,经G418筛选,获得阳性细胞克隆,扩增后分别用逆转录聚合酶链反应(RT-PCR)、免疫印迹(Western-blotting)检测t-PA转录和蛋白表达水平,用底物发色法检测t-PA的活性。逆转录聚合酶链反应检测出了t-PA的稳定转染细胞克隆,免疫印迹证实该克隆细胞有含Myc标签的t-PA蛋白表达,底物发色法检测发现其表达产物的活性增加。结果证明成功建立了稳定表达t-PA基因的人脐静脉内皮细胞株。     

新型酪氨酸激酶抑制剂XCF-43b体外抗血管新生研究

目的 探究XCF-43b 体外抗血管新生机理.方法 鸡胚尿囊膜(CAM)检测化合物抑制血管新生能力,MTT检测其对HUVEC细胞增殖影响,划痕实验和微管形成实验检测化合物对HUVEC迁移和微管形成的影响,Western印迹检测VEGFR2信号通路相关蛋白表达情况.结果 XCF-43b能够抑制CAM血管新生,抑制HUVEC细胞增殖的IC50为(28.42±7.23) μmol/L,对在VEGF刺激下的HUVEC的IC50值为(9.03±1.28) μmol/L,此外,2.5 μmol/L的XCF-43b能够抑制HUVEC细胞的迁移和微管形成;且能够抑制VEGFR2及其下游信号因子的激活.结论 XCF-43b通过抑制VEGFR2信号通路来抑制血管新生.     

新生血管特异抗体的制备及生物活性鉴定

目的 研制识别肿瘤新生血管的单克隆抗体及其活性。方法 用肝癌细胞条件培养基刺激人血管内皮细胞，以此作为抗原免疫小鼠制备抗体。用免疫组化、ELISA和流式细胞术筛选肿瘤血管特异抗体并鉴定其免疫特性。用MTT法研究抗体对血管内皮细胞增殖的影响。结果 抗体AA98选择性地识别肿瘤血管和人血管内皮细胞，抑制血管内皮细胞的增殖。结论 抗体AA98在肿瘤诊断和治疗方面具有潜在的应用价值。     

Human RCAN3 gene expression and cell growth in endothelial cells

Regulator of calcineurin 3 (RCAN3) belongs to the human RCAN gene family, which also includes RCAN1 and RCAN2. All three members interact with and inhibit calcineurin. Based on this effect, several studies have demonstrated a role for RCAN1 and RCAN2 on inflammation, using human umbilical vein endothelial cells (HUVECs) as a model. RCAN1 and 2 are strongly induced by vascular endothelial growth factor (VEGF), inhibit cell proliferation and down-regulate many pro-inflammatory and pro-angiogenic genes. The present work is the first study to investigate the role of RCAN3 on inflammation in HUVECs. RCAN3 isoforms have been characterized and quantified in HUVECs; only those with the same frame are expressed and show a peculiar expression pattern. RCAN3 inhibits HUVEC proliferation both basally and under VEGF or phorbol 12-myristate 13-acetate-stimulated conditions, however it does not modulate gene expression of the chosen inflammatory genes. Results indicate an interesting role for RCAN3 in modulating HUVEC proliferation, independently from the inflammatory and angiogenic processes.