血管内皮生长因子受体信号转导通路与肿瘤血管生成

血管内皮生长因子是促进血管生成的重要调节因子.它能促进内皮细胞增殖、迁移,阻止内皮细胞凋亡、管腔网状结构退化,增加血管渗透性.所有这些作用都是通过血管内皮生长因子受体信号转导通路实现的.它们在肿瘤血管生成、肿瘤生长中起着重要的作用.以血管内皮生长因子受体信号转导通路为靶点是开发肿瘤血管生成抑制剂的理想策略.     

血管内皮细胞生长因子研究进展

从不同侧面阐述了血管内皮细胞生长因子（VEGF）在新生血管形成中的作用.VEGF诱导新生血管形成,具有血管渗透性,是新生血管形成的主要调控者之一.VEGF mRNA不同剪接,形成5种VEGF变异体(isoform)即VEGF121-206.VEGF诱导新生血管的调控过程、拮抗VEGF成为大家竞相研究的领域.     

杀伤血管内皮生长因子受体 1 阳性细胞的靶向毒素

白喉毒素 (diphtheria toxin DT) 是棒状白喉杆菌被β噬菌体感染后分泌的一种外毒素. 它可以阻断真核细胞的蛋白质合成,杀死细胞. 血管内皮生长因子 (VEGF) 的 R82A, K84A, H86A 突变体可以和肿瘤血管上高表达的 VEGF 受体 1 (VEGFR-1) 特异性结合. 首先从白喉杆菌中提取基因组 DNA,扩增出白喉毒素 C 区、 T 区基因. 并运用点突变技术,制成 VEGF 的 R82A, K84A, H86A 突变体. 利用这个可以和肿瘤血管上特异性受体相结合的 VEGF 的突变体,代替白喉毒素上的受体结合区,制成了针对 VEGFR-1 的靶向融合毒素——— DT391-mVEGF. 以去除了受体结合区的 DT391 为阴性对照,细胞实验表明,融合毒素对 VEGFR-1 阳性的肿瘤细胞有特异性杀伤作用.     

利用酵母双杂交系统研究人血管内皮生长因子受体Flt-1的配体结合域

The vascular endothelial growth factor (VEGF) receptor Flt-1 is charaterized by seven Ig-like loops within the extracellular domain. To identify which part is responsible for ligand binding, four cDNA clones coding for truncated Flt-1 mutants consisting of loop 1, 1-2, 2-3 and 1-3 were obtained by PCR from human cardiac cDNA library and inserted into the vectors of the yeast two-hybrid system, with VEGF cDNA on the partner plasmid. The paired plasmids were transformed into yeast strain SFY526, and tested by filter membrane method and β-galactosidase activity. The results showed that Flt-1(1-2)、Flt-1(2-3) and Flt-1(1-3) all were able to bind VEGF, of which Flt-1(1-3) showed the highest binding affinity, but no binding of VEGF was observed with Flt-1(2) and VEGF.     

白介素-1受体拮抗剂抑制角膜新生血管及血管内皮生长因子表达的研究

为了探讨白介素-1受体拮抗剂(interleukin 1 receptor antagonist,IL_1ra)对大鼠角膜新生血管(corneal neovascularization,CNV)中血管内皮生长因子(vascular endothelial growth factor,VEGF)表达的影响及其对CNV生长的作用,采用角膜缝线法建立大鼠CNV模型,分成正常对照组、单纯角膜缝线组和缝线加IL_1ra结膜下注射组三组,于术后1w、2w分别计算各组CNV面积,观察CNV生长情况;术后1w各组随机处死4只大鼠,取角膜组织采用RT_PCR检测VEGF的表达。结果表明,正常对照组无CNV生长,VEGF低表达;单纯缝线组CNV生长旺盛,VEGF高表达,CNV面积、VEGF mRNA相对吸光度值与其他两组相比有极显著差异(p<0.01);IL_1ra组CNV生长稀疏,VEGF表达较低,但仍高于正常对照组,差异有统计学意义(p<0.01)。     

乳腺癌组织中转化生长因子-beta1 和血管内皮生长因子的表达 及其与血管生成的关系

目的:研究转化生长因子-β1(transforming growth factor-β1,TGF-β1)和血管内皮生长因子(vascular endothelial cell growth factor,VEGF)在乳腺癌组织中的表达及其与血管生成的关系。方法:选取65例手术切除乳腺癌蜡块标本及其周围正常乳腺组织,分为两组:A组为对照组,检测标本为乳腺癌癌旁正常乳腺组织;B组为实验组,检测标本为乳腺癌组织,采用免疫组织化学染色和形态计量检测TGF-β1和VEGF在乳腺癌组织中的表达。利用CD34相关抗原标记血管内皮细胞,计数微血管密度(intratumoral mier oveseulardensity,MVD),并分析其与TGF-β1和VEGF表达的关系。结果:65例乳腺癌组织中,TGF-β1的阳性表达率为69.23%(45/65),TGF-β1阳性表达者MVD值(25.31±4.05)显著高于TGF-β1阴性表达者(21.23±4.29);VEGF的阳性表达率为78.46%(51/65),VEGF阳性表达者MVD值(26.62±3.41)亦明显显著高于VEGF阴性表达者(18.95±6.52)(均P<0.05)。不同病理类型的乳腺癌组织中TGF-β1、VEGF的阳性表达率比较差异无统计学意义(P>0.05),但TGF-β1、VEGF的阳性表达与乳腺癌的组织分级、淋巴结转移呈显著正相关(均P<0.05),且组织学分级越高、淋巴结转移越多,MVD值越大。结论:TGF-β1与VEGF在乳腺癌组织的表达高于正常乳腺组织,并与乳腺癌肿瘤血管的生成有关,二者有望作为乳腺癌恶性程度、浸润转移等生物学行为的评估指标。     

血管内皮生长因子受体1酪氨酸激酶的克隆、表达和功能

血管内皮生长因子受体 1(Flt 1)在血管生成过程中起着重要的作用。Flt 1胞内域的酪氨酸激酶直接参与了VEGF与Flt 1结合后的胞内信号转导途径。在原核系统中表达得到具有酶活性的Flt 1酪氨酸激酶融合蛋白 ,并进行了初步的性质研究。利用逆转录PCR技术从人肝癌组织总RNA中得到Flt 1酪氨酸激酶区的cDNA ,将其克隆到表达载体质粒 pGEX KG中 ,并在大肠杆菌BL2 1(DE3) pLysS中表达、纯化 ,得到可溶的Flt 1酪氨酸激酶融合蛋白 (GST F)。虽然GST F不包含目前已知的磷酸化位点 ,但研究表明GST F能够进行自磷酸化反应 ,并且其活性需要镁离子或锰离子的参与。同时发现GST F能够磷酸化合成底物 polyE4Y ,而不能作用于MBP和Src相关肽。底物磷酸化时最适的镁离子和锰离子浓度分别为 15mmol/L和 0 .5mmol/L。GST F为寻找抗肿瘤药物提供了一个有效工具     

血管内皮细胞生长因子受体Flt—1胞外区基因的克隆及其在中国仓？…

朋原代培养的人脐静脉血管内皮细胞（ＨＵＶＥＣ）提取细胞总ＲＮＡ,采用逆转录ＰＣＲ（ＲＴ－ＰＣＲ）方法得到ＶＥＧＦ受体Ｆｌｔ－１胞外区前３个ＩｇＧ样区域ｃＤＮＡ片段（Ｆｌｔ－１ｎ３）。将获得的受体基因克隆到真核表达载体ｐｃＤ－ＮＡ３．１中,得到重组质粒ｐｃＤＮＡ３．１／Ｆｌｔ－１ｎ３,通过南体转染方法将其转入中国仓鼠卵巢细胞（ＣＨＯ）,用Ｇ４１８筛选得到稳定表达目的蛋白的细胞砍隆。经固相结合实验筛选     

血管内皮标记物及血管内皮生长因子在胸水细胞中的表达和意义

探讨CD31、CD34、CD105及VEGF在胸水中的表达.应用免疫细胞化学染色,免疫荧光染色,Western-blot技术检测200例非小细胞肺癌患者胸水、30例增生胸水和20例炎性胸水中CD31、CD34、CD105及VEGF的表达.CD31、CD34、CD105及VEGF在非小细胞肺癌胸水中的表达量明显高于增生和炎性胸水表达(P＜0.05).非小细胞肺癌胸水患者CD31、CD34、CD105及VEGF高表达,并且在存在着肿瘤细胞血管样管型结构中表达量明显高于未发现肿瘤细胞血管样管型的胸腔积液.检测CD31、CD34、CD105及VEGF在胸腔积液中的表达情况可能对判断患者的预后有一定价值.     

PEG-PEI共聚物介导VEGF165基因转染及对内皮细胞生长的影响

为了考察PEG-PEI共聚物作为基因载体介导VEGF165基因的能力,合成不同接枝量的PEG-PEI共聚物,考察共聚物的细胞毒性,同时采用PCR技术获得上下游含有HindⅢ和BamHⅠ酶切位点的目的基因VEGF165,与pEGFP-C1构建重组质粒pEGFP-VEGF165,将PEG-PEI作为基因载体,与pEGFP-VEGF165通过自组装成DNA复合物,使其转染脐静脉内皮细胞(HUVEc),测定发荧光细胞百分数获得转染率,利用ELISA、RT-PCR检测VEGF的表达,用MTT法考察VEGF165转染HUVEc后对内皮细胞生长的影响.结果显示,形成PEG-PEI共聚物后可显著降低PEI的细胞毒性.作为基因载体介导pEGFP-VEGF165转染HUVEc后,在荧光显微镜下可见强绿色荧光蛋白表达,转染率与接枝PEG的量及N/P有关,PEG-PEI(5-25-1)在N/P=30时转染率达到最大值,比PEI显著提高.转染后血管内皮生长因子(VEGF)蛋白表达及mRNA水平均有显著提高,且可有效地刺激内皮细胞增殖.研究表明,PEG-PEI共聚物可做为基因载体,有效地介导pEGFP-VEGF165基因的传递.     

小鼠天然可溶性VEGF受体基因sflt-1的克隆及鉴定

 可溶性血管内皮细胞生长因子受体 (sFlt 1 )与膜表面受体Flt 1竞争结合血管内皮细胞生长因子 (VEGF) ,并且与膜表面受体Flt 1及KDR形成异源二聚体 .完全阻断VEGF的生物学活性 ,除与sFlt 1的结合部位结构域有关外 ,还与整个蛋白质分子的高效分泌表达有关 .而蛋白质分子的高效分泌表达与蛋白质在细胞内高尔基体及内质网的加工密切相关 ,基因工程重组可溶性受体由于一些尚未明了的原因 ,往往不能高效表达 ,从而大大影响了其应用价值 .利用RT PCR技术从胚胎小鼠中扩增出天然可溶性VEGF受体基因sflt 1 ,克隆于pcDNA3载体中 ,在COS 7细胞中短暂表达 ;并克隆至pET42b载体中 ,经IPTG诱导后 ,可大量稳定表达与His Tag形成的融合蛋白 ,经HisNi柱纯化 ,可特异性结合VEGF .可溶性受体sFlt 1在肿瘤组织的高效表达可有效阻断新生血管的形成 ,从而为肿瘤的治疗探索一种方法 .     

血管内皮生长因子及其受体在斑马鱼胚胎血管发育中的作用

血管内皮生长因子(vascular endothelial growth factor,VEGF)是一种多功能的细胞因子,其主要作用是促进血管内皮细胞增殖和增加血管通透性,是肿瘤及正常组织血管生成的中心调控因素。以VEGF为靶点的肿瘤血管靶向性治疗成为近几年肿瘤治疗的新途径。斑马鱼作为一种重要的模式生物,被广泛用于胚胎的分子发育机制、疾病模型的构建以及药物筛选等研究中。文章对斑马鱼作为心血管系统研究模型的优势及其血管研究方法做一阐述,重点对斑马鱼VEGF及其受体的最新研究进展做了介绍,并展望了其发展前景。     

肿瘤饥饿疗法的新靶标——内分泌腺衍生血管内皮生长因子

“肿瘤饥饿疗法”是通过抑制促肿瘤血管新生细胞因子的作用,阻断肿瘤血管形成,最终实现“饿死”肿瘤细胞的一种治疗方法．内分泌腺衍生血管内皮生长因子(EG-VEGF)是在2001年被发现的一个组织选择性促血管新生因子．近年来的研究表明,EG-VEGF还兼有促进造血干细胞分化、刺激胃肠道收缩及影响肠神经系统发育等多种生理功能．EG-VEGF的异常表达与多种肿瘤及血管新生依赖性疾病的发生发展密切相关,有望作为相应的治疗靶点开发诊断及治疗试剂．本文对有关研究进展及应用前景作一简要综述．     

Regulation of Vascular Endothelial Growth Factor Receptor-2 (Flk-1) Expression in Vascular Endothelial Cells

We have previously reported the existence of a synergistic interaction between vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in the induction of angiogenesisin vitro.Here we demonstrate that bFGF increases VEGF receptor-2 (VEGFR-2/Flk-1) expression: mRNA levels were increased by 4.5- to 8.0-fold and total protein by 2.0- to 3.5-fold, in bovine microvascular endothelial (BME), aortic endothelial (BAE), and transformed fetal aortic (GM7373) endothelial cells. VEGF itself did not affect VEGFR-2 expression, and neither bFGF nor VEGF altered expression of FGF receptor-1. We also show that synergism occurs at the level of proliferation when this is measured in a three-dimensional but not in a conventional two-dimensional assay. Differences in the level of VEGFR-2 expression were also observed when cells were grown on or within collagen gels under different conditions: mRNA levels were lowest under sparse conditions, increased 20- to 26-fold at confluence, and increased even further (57-fold) when cells were cultured in suspension in three-dimensional collagen gels. Finally, a synergistic increase was seen in the level of expression of urokinase and urokinase receptor mRNAs when cells were exposed to bFGF and VEGF for 4 days. These findings demonstrate that the level of VEGFR-2 expression can be modulated by environmental factors including cytokines and the geometry of the culture conditions and provide some insight into the mechanisms of synergism between bFGF and VEGF in the induction of angiogenesisin vitro.     

活血化瘀方对糖尿病模型大鼠糖脂代谢、血管内皮生长因子和血管紧张素II1型受体表达的影响

目的:探讨活血化瘀方对糖尿病模型大鼠糖脂代谢、血管内皮生长因子(VEGF)和血管紧张素Ⅱ1型受体(AT1R)表达的影响。方法:选取健康雄性SD大鼠50只,适应性喂养7 d后以随机数字表法分成对照组10只、模型组13只、中药组14只、西药组13只。其中模型组与对照组予以纯净水灌胃,中药组予以活血化瘀通络中药配方颗粒灌胃,西药组则予以厄贝沙坦灌胃,1次/d,连续灌胃16周。分别比较各组大鼠的糖脂代谢指标水平及24 h尿蛋白定量、糖化血红蛋白、血清肌酐水平,并检测肾组织VEGF和AT1R表达情况。结果:模型组、中药组、西药组大鼠空腹血糖(FBG)、总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)水平均高于对照组,中药组、西药组大鼠LDL-C水平低于模型组,中药组大鼠FBG水平低于模型组与西药组(P0.05)。模型组、中药组、西药组大鼠24 h尿蛋白定量与糖化血红蛋白均高于对照组,中药组、西药组大鼠24 h尿蛋白定量低于模型组(P0.05)。模型组、中药组、西药组大鼠VEGF、AT1R水平均高于对照组,中药组、西药组大鼠VEGF、AT1R水平低于模型组,中药组大鼠AT1R水平低于西药组(P0.05)。结论:活血化瘀方可有效改善糖尿病大鼠糖脂代谢状态,通过抑制VEGF与AT1R的表达水平,延缓糖尿病的发生与发展。     

Synergistic Binding of Vascular Endothelial Growth Factor-A and Its Receptors to Heparin Selectively Modulates Complex Affinity

Angiogenesis is a highly regulated process orchestrated by the VEGF system. Heparin/heparan sulfate proteoglycans and neuropilin-1 (NRP-1) have been identified as co-receptors, yet the mechanisms of action have not been fully defined. In the present study, we characterized molecular interactions between receptors and co-receptors, using surface plasmon resonance and in vitro binding assays. Additionally, we demonstrate that these binding events are relevant to VEGF activity within endothelial cells. We defined interactions and structural requirements for heparin/HS interactions with VEGF receptor (VEGFR)-1, NRP-1, and VEGF₁₆₅ in complex with VEGFR-2 and NRP-1. We demonstrate that these structural requirements are distinct for each interaction. We further show that VEGF₁₆₅, VEGFR-2, and monomeric NRP-1 bind weakly to heparin alone yet show synergistic binding to heparin when presented together in various combinations. This synergistic binding appears to translate to alterations in VEGF signaling in endothelial cells. We found that soluble NRP-1 increases VEGF binding and activation of VEGFR-2 and ERK1/2 in endothelial cells and that these effects require sulfated HS. These data suggest that the presence of HS/heparin and NRP-1 may dictate the specific receptor type activated by VEGF and ultimately determine the biological output of the system. The ability of co-receptors to fine-tune VEGF responsiveness suggests the possibility that VEGF-mediated angiogenesis can be selectively stimulated or inhibited by targeting HS/heparin and NRP-1.     

血管内皮生长因子受体-2所介导信号通路的研究进展

血管新生是许多生理和病理进程发生的重要机理．在生物体内,血管新生需经过多步精细调控历程,现有研究表明,血管内皮生长因子(VEGF)及其受体蛋白酪氨酸激酶,尤其是血管内皮生长因子受体-2(VEGFR-2)所介导的信号级联通路是其中关键性的调节途径．VEGF/VEGFR-2所介导的信号级联通路可以调控血管内皮细胞的增殖、迁移、存活和通透性的改变,促进血管的新生．VEGF与VEGFR-2的胞外区特异性结合后,引起受体的二聚化和自身的交互磷酸化,使胞内特定的酪氨酸残基磷酸化．下游信号蛋白可以通过其Src同源结构域-2(SH2)与VEGFR-2结合,随后激活下游的效应蛋白,调控内皮细胞的生物学活性．此外,VEGF/VEGFR-2信号通路还可以下调树突细胞(DC)的活性．对VEGF/VEGFR-2信号通路作用的深入了解,将有助于新药的研发．     

A Polysaccharide Isolated from the Medicinal Herb Bletilla striata Induces Endothelial Cells Proliferation and Vascular Endothelial Growth Factor Expression in vitro

A polysaccharide from the traditional Chinese medicinal herb, Bletilla striata (Thunb.) Reichb. f., was isolated, purified and characterized. It induced the proliferation of human umbilical vascular endothelial cells and the expression of vascular endothelial growth factor up to 156% and 147% of control after 72 h, respectively. Revisions requested 8 November 2005; Revisions received 13 January 2006     

Endothelial Heparan Sulfate 6-O-Sulfation Levels Regulate Angiogenic Responses of Endothelial Cells to Fibroblast Growth Factor 2 and Vascular Endothelial Growth Factor

Fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor 165 (VEGF₁₆₅) are potent pro-angiogenic growth factors that play a pivotal role in tumor angiogenesis. The activity of these growth factors is regulated by heparan sulfate (HS), which is essential for the formation of FGF2/FGF receptor (FGFR) and VEGF₁₆₅/VEGF receptor signaling complexes. However, the structural characteristics of HS that determine activation or inhibition of such complexes are only partially defined. Here we show that ovarian tumor endothelium displays high levels of HS sequences that harbor glucosamine 6-O-sulfates when compared with normal ovarian vasculature where these sequences are also detected in perivascular area. Reduced HS 6-O-sulfotransferase 1 (HS6ST-1) or 6-O-sulfotransferase 2 (HS6ST-2) expression in endothelial cells impacts upon the prevalence of HS 6-O-sulfate moieties in HS sequences, which consist of repeating short, highly sulfated S domains interspersed by transitional N-acetylated/N-sulfated domains. 1–40% reduction in 6-O-sulfates significantly compromises FGF2- and VEGF₁₆₅-induced endothelial cell sprouting and tube formation in vitro and FGF2-dependent angiogenesis in vivo. Moreover, HS on wild-type neighboring endothelial or smooth muscle cells fails to restore endothelial cell sprouting and tube formation. The affinity of FGF2 for HS with reduced 6-O-sulfation is preserved, although FGFR1 activation is inhibited correlating with reduced receptor internalization. These data show that 6-O-sulfate moieties in endothelial HS are of major importance in regulating FGF2- and VEGF₁₆₅-dependent endothelial cell functions in vitro and in vivo and highlight HS6ST-1 and HS6ST-2 as potential targets of novel antiangiogenic agents.