VEGF165过表达对小鼠乳腺癌及肿瘤周边皮肤血管生成模式的影响研究

已证实乳腺癌细胞表达有VEGF165的第3个受体NRP1,但目前在乳腺癌细胞中过表达VEGF165对肿瘤周围血管生成模式的影响尚未有人研究.通过脂质体转染的方法得到稳定转染过表达VEGF165的细胞克隆,皮内接种研究其对周围皮肤生血管模式的影响.结果显示VEGF165在乳腺癌中过表达可使肿瘤周围皮肤产生树状血管生成模式,且会诱导不成熟的血管生成并致出血.稳定过表达VEGF165的乳腺癌克隆的建立,为进一步研究过表达VEGF165对乳腺癌的影响奠定了基础.     

雌激素刺激脑血管内皮细胞分泌VEGF及新血管形成

为了探讨雌激素对脑血管形成的影响,本实验利用体外培养的种植在matrigel上的脑血管内皮细胞作为模型,对雌激素刺激形成的新生血管进行照相记录,计算机软件分析.结果显示:雌激素呈剂量依赖性地刺激培养的脑血管内皮细胞分泌VEGF和新血管形成,而雄激素对培养的脑血管内皮细胞新血管形成没有任何影响;VEGF可以显著地促进内皮细胞新血管的形成.以上结果说明,雌激素通过刺激VEGF的分泌作用进而促进脑血管内皮细胞新血管的形成.     

电刺激小脑顶核促脑缺血后血管内皮生长因子表达的意义

探讨电刺激小脑顶核(FNS)对局部脑缺血后血管内皮生长因子(VEGF)表达和毛细血管新生的影响．以线栓法制成大鼠右侧大脑中动脉梗塞模型(MCAO)，大鼠随机分为假手术对照组、MCAO组、电刺激小脑顶(MCAO FNS)干预组，以免疫组织化学法检测VEGF、内皮细胞阳性表达及毛细血管记数，大脑中动脉梗塞后，缺血区神经元变性、坏死，VEGF、内皮细胞在半暗带有少量表达，毛细血管数较对照组增加，经电刺激小脑顶核干预后，VEGF、内皮细胞大量表达，毛细血管数目明显增加，有统计学差异．电刺激小脑顶核可通过促VEGF表达、内皮细胞增殖，从而促进毛细血管新生。     

内皮细胞抑制素在毕节酵母中的表达与活性分析

采用PCR技术从人胎脑cDNA库中扩增了人内皮细胞抑制素基因。经DNA序列分析后将扩增的基因克隆至酵母载体pPICgK，获得的重组质粒pPIC9K-EDN转化毕节酵母GSll5，构建表达内皮细胞抑制素的酵母工程菌P.pastoris GSll5(pPIC9K-EDN)。SDS-PAGE分析结果显示：人内皮细胞抑制素在重组酵母GSll5(pPIC9K-EDN)中获得高效表达。用30L发酵罐构对建的工程菌进行高密度发酵，经甲醇诱导48h，生物量达到250OD，分泌量为150mg/L，发酵液经SP Streamline，SP Sepharose FF阳离子交换柱和SephamseHeparin Hi Trap柱纯化，产物纯度达到98％。纯化产物具有免疫活性并能抑制bFGF诱导的鸡胚绒毛尿囊膜血管生成。     

重组人血管内皮生长因子165在大肠杆菌中的可溶性表达、纯化与体外活性评价

人血管内皮生长因子165(VEGF165)可有效促进血管新生和增加血管通透性,在伤口愈合方面有重要医疗价值。建立获取高纯度、高活性的优质重组VEGF165蛋白的方法具有重要意义。研究利用带有6组氨酸标签的二硫键形成蛋白A(Dsb A)的E.coli表达系统实现了Dsb A-VEGF165融合蛋白的可溶性表达;诱导过程中添加5%(v/v)的乙醇可显著提高工程菌中可溶性融合蛋白表达水平。融合蛋白通过Ni亲和层析粗纯,并经牛肠激酶酶切去除标签蛋白。随后利用肝素亲和层析精纯获得重组人VEGF165蛋白。非还原及还原SDS-PAGE电泳检测到分子量为约40 k Da的同源二聚体蛋白,促HUVEC细胞增殖实验显示重组蛋白具有较优的活性,EC50为13 ng/m L。研究实现了Dsb A-VEGF165的在E.coli中可溶性表达,建立了经济、高效的纯化方法,获得了高质量、高活性的重组人VEGF165蛋白。     

血管内皮生长因子(VEGF)与肝再生

血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)能特异地直接作用于血管内皮细胞,刺激血管内皮细胞的分裂、增殖并诱导血管的形成.它通过与血管内皮细胞的受体结合发挥作用.肝受到各种损伤包括部分肝切除后,肝再生就得以启动.在肝再生过程中,VEGF对内皮细胞的生长增殖起有效促进作用,并且能诱导组织胶原酶、血纤维蛋白酶原的激活,增加血管的通透性,这对血管再生起着极其重要的作用.而血管再生是肝再生的一个重要组成部分,它不仅能给肝细胞提供血液支持,而且能促进肝脏结构的重构.因此VEGF在肝再生过程中具有重要的作用.     

血管内皮生长因子在大鼠乳腺炎中的作用

应用组织化学方法及免疫组化技术对正常组和急性攻毒组大鼠乳腺组织血管内皮生长因子（Vascular Endothelial Growth Factor,VEGF）的表达水平、肥大细胞的活力和炎性细胞的动态变化进行了研究.结果表明,急性攻毒后6 h乳腺组织VEGF在腺泡间、小叶间质、血管周围及部分肥大细胞内的表达出现一个峰值,而后表达开始减弱,攻毒48 h后表达又迅速增强;攻毒组大鼠的肥大细胞及其脱颗粒和炎性细胞随着攻毒时间的延长急剧增多,均极显著高于正常组（P〈0.01）,且其变化趋势均与VEGF表达趋势相吻合;同时,小叶间质内的小血管内皮细胞开始出现裂隙、脱落损伤.由此表明,VEGF和肥大细胞参与大鼠乳腺炎的发病过程.     

VEGF在妇产科及生殖领域中的应用

血管内皮生长因子(VEGF)是一组功能强大且能产生多种生物学效应的细胞因子,它能够促进血管内皮细胞增殖及体内新生血管形成并增加血管通透性.近年来在女性生殖内分泌领域中VEGF受到相当的重视.本研究就VEGF的生物特性,以及其在妇产科领域、辅助生殖技术中的应用作一综述.     

桦树皮提取物抑制血管生成作用的研究

研究桦树皮提取物对血管生成的抑制作用.应用MTT法检测桦树皮提取物对人脐静脉内皮细胞的增殖抑制作用,采用Realtime PCR方法检测桦树皮提取物对人脐静脉内皮细胞中VEGFmRNA表达的影响,应用ELISA测定细胞上清液中VEGF的分泌情况.实验表明,桦树皮提取物能显著抑制人脐静脉内皮细胞增殖,最高浓度150 μg/mL其抑制率达48.4%,能明显抑制人脐静脉内皮细胞内VEGFmRNA的表达及VEGF分泌.结果表明桦树皮提取物可通过抑制细胞内VEGF mRNA的表达及VEGF的分泌有效抑制人脐静脉内皮细胞的增殖.     

膝关节镜下所见血管翳患者滑膜中VEGF、TGF-β_1的表达及其意义

为探讨VEGF、TGF-β1两因子与血管翳的相互关系,为血管翳的临床治疗提供指导,选择行膝关节镜手术且发现在1~3个部位出现血管翳的患者,根据血管翳出现部位的数量将其滑膜标本筛选分为3个实验组,每组15例,其中未发现血管翳患者滑膜的标本为对照组。采用免疫组化技术检测与观察VEGF、TGF-β1在各组中的表达,分析其表达意义。结果表明:与对照组相比,实验组中的VEGF、TGF-β1呈高表达,差异有统计学意义(P0.05),且与血管翳出现的部位数相关,随着出现部位的增加,VEGF、TGF-β1的阳性表达率也随之增高;将VEGF与TGF-β1的检验结果进行Spearman相关分析,结果显示两因子的阳性率呈正相关(r=0.910,P=0.000)。这表明,VEGF、TGF-β1的过度表达与膝关节血管翳的形成密切相关,检测膝关节滑膜组织中VEGF和TGF-β1的表达有助于预测血管翳的发展趋势。     

Notch-dependent VEGFR3 upregulation allows angiogenesis without VEGF-VEGFR2 signalling

Developing tissues and growing tumours produce vascular endothelial growth factors (VEGFs), leading to the activation of the corresponding receptors in endothelial cells. The resultant angiogenic expansion of the local vasculature can promote physiological and pathological growth processes. Previous work has uncovered that the VEGF and Notch pathways are tightly linked. Signalling triggered by VEGF-A (also known as VEGF) has been shown to induce expression of the Notch ligand DLL4 in angiogenic vessels and, most prominently, in the tip of endothelial sprouts. DLL4 activates Notch in adjacent cells, which suppresses the expression of VEGF receptors and thereby restrains endothelial sprouting and proliferation. Here we show, by using inducible loss-of-function genetics in combination with inhibitors in vivo, that DLL4 protein expression in retinal tip cells is only weakly modulated by VEGFR2 signalling. Surprisingly, Notch inhibition also had no significant impact on VEGFR2 expression and induced deregulated endothelial sprouting and proliferation even in the absence of VEGFR2, which is the most important VEGF-A receptor and is considered to be indispensable for these processes. By contrast, VEGFR3, the main receptor for VEGF-C, was strongly modulated by Notch. VEGFR3 kinase-activity inhibitors but not ligand-blocking antibodies suppressed the sprouting of endothelial cells that had low Notch signalling activity. Our results establish that VEGFR2 and VEGFR3 are regulated in a highly differential manner by Notch. We propose that successful anti-angiogenic targeting of these receptors and their ligands will strongly depend on the status of endothelial Notch signalling.     

Homoharringtonine induces apoptosis of endothelium and down－regulates VEGF expression of K562 cells

Homoharringtonine (HHT) has currently been used successfully in the treatment of acute and chronic myeloid leukemias and has been shown to induce apoptosis of different types of leukemic cells in vitro. Emerging evidence suggests that angiogenesis may play an important role in hematological malignancies, such as leukemia. However, whether HHT can relieve leukemia by anti-angiogenesis is still unknown. We investigated the anti-angiogenesis potential of HHT with the human umbilical vein endothelial cell line (ECV304) and leukemic cell line (K562) in vitro. Cellular proliferation was determined by MTT assay and apoptosis was analyzed by flow cytometry, The mRNA expression of vascular endothelial growth factor (VEGF) was assessed by RT-PCR and VEGF protein production was detected by Western blot. Inhibition of cell proliferation and induction of apoptosis by HHT were discovered in ECV304 cells, and appeared in a dose- and time-dependent manner, Also, treatment with HHT caused down-regulation of VEGF mRNA expression in K562 cells in similar dose- and time-dependent manner and inhibition of VEGF protein production in K562 cells in response to the enhancing concentration of HHT. The results demonstrated that HHT could also induce apoptosis in endothelium and down-regulate VEGF expression in K562 cells. In conclusion, we believe HHT has anti-angiogenesis potential and speculate that HHT might exert its anti-leukemia effects via reduction of angiogenesis.     

Pathophysiological consequences of VEGF-induced vascular permeability

Although vascular endothelial growth factor (VEGF) induces angiogenesis, it also disrupts vascular barrier function in diseased tissues. Accordingly, VEGF expression in cancer and ischaemic disease has unexpected pathophysiological consequences. By uncoupling endothelial cell-cell junctions VEGF causes vascular permeability and oedema, resulting in extensive injury to ischaemic tissues after stroke or myocardial infarction. In cancer, VEGF-mediated disruption of the vascular barrier may potentiate tumour cell extravasation, leading to widespread metastatic disease. Therefore, by blocking the vascular permeability promoting effects of VEGF it may be feasible to reduce tissue injury after ischaemic disease and minimize the invasive properties of circulating tumour cells.     

Anti-angiogenesis effect of generation 4 polyamidoamine/vascular endothelial growth factor antisense oligodeoxynucleotide on breast cancer in vitro

Objective: To study the effects of the generation 4 polyamidoamine/vascular endothelial growth factor antisense oligodeoxynucleotide (G4PAMAM/VEGFASODN) compound on the expressions of vascular endothelial growth factor (VEGF) and its mRNA of breast cancer cells and on the inhibition of vascular endothelial cells. Methods: We examined the morphology of G4PAMAM/VEGFASODN compound and its pH stability, in vitro transfection efficiency and toxicity, and the expressions of VEGF and its mRNA. Methyl thiazolyl tetrazolium assay was used to detect the inhibitory function of the compound on vascular endothelial cells. Results: The compound was about 10 nm in diameter and was homogeneously netlike. From pH 5 to 10, it showed quite a buffered ability. The 48-h transfection rate in the charge ratio of 1:40 was 98.76%, significantly higher than that of the liposome group (P<0.05). None of the transfection products showed obvious toxicity on the cells. The expressions of both VEGF protein and its mRNA after G4PAMAM/VEGFASODN transfection decreased markedly. Conclusion: With a low toxicity, high safety, and high transfection rate, G4PAMAM/VEGFASODN could be a promising gene vector. Specifically, it inhibits VEGF gene expression efficiently, laying a basis for further in vivo animal studies.     

Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo.

Clinical and experimental studies suggest that angiogenesis is a prerequisite for solid tumour growth. Several growth factors with mitogenic or chemotactic activity for endothelial cells in vitro have been described, but it is not known whether these mediate tumour vascularization in vivo. Glioblastoma, the most common and most malignant brain tumour in humans, is distinguished from astrocytoma by the presence of necroses and vascular proliferations. Here we show that expression of an endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF), is induced in astrocytoma cells but is dramatically upregulated in two apparently different subsets of glioblastoma cells. The high-affinity tyrosine kinase receptor for VEGF, flt, although not expressed in normal brain endothelium, is upregulated in tumour endothelial cells in vivo. These observations strongly support the concept that tumour angiogenesis is regulated by paracrine mechanisms and identify VEGF as a potential tumour angiogenesis factor in vivo.     

红景天苷上调大鼠心肌梗死后心肌组织VEGF的表达

目的:探讨红景天苷对大鼠心肌梗死后心肌组织血管内皮生长因子(VEGF)的表达调控作用及其可能机制。方法:建立大鼠心梗模型后,随机分为模型组、红景天苷3个不同剂量组,每组8只大鼠,另设假手术组8只。术后48 h,红景天苷组分别给予低、中、高,即10,20,40 mg·(kg·d)-1灌胃;模型组和假手术组给予生理盐水20 ml·(kg·d)-1灌胃。4周后处死大鼠,取大鼠心肌组织,反转录PCR(RT-PCR)法测定VEGF mRNA的表达。应用免疫组化和免疫印迹法分析左心室心肌组织VEGF蛋白的表达情况。结果:RT-PCR结果表明,和模型组相比,红景天苷各剂量组VEGF mRNA的表达均明显升高(P<0.01)。免疫组化和免疫印迹分析结果表明,和模型组相比,红景天苷各剂量组心肌组织胞浆中VEGF蛋白的表达均显著升高(P<0.01)。结论:红景天苷通过上调VEGF的表达而促大鼠心肌梗死后心肌组织的血管新生。     

缺氧预适应小鼠海马脑区VEGF蛋白表达增加

目的 观察重复低氧对小鼠海马组织内血管内皮生长因子(vascular endothelial growth factor,VEGF) 蛋白质表达的影响.方法 小鼠随机分为3组,分别暴露低氧0次(H0),1次(H1),4 次(H4)后取海马组织,应用 Western Blot 技术,检测小鼠海马组织内VEGF的蛋白表达变化.结果 实验发现,H0,H1和H4组海马组织内VEGF均表达,H1组与H0组之间没有差异,H4组显著升高（P<0.05,vs. H0 and H1）.结论 VEGF在急性低氧预适应小鼠海马组织表达增加可能参与预适应的形成及脑保护.     

6个氨基酸小C肽人胰岛素原类似物基因的构建和表达

用片段置换法，从在Ｃ肽两端具有酶切位点的双突人胰岛素原因中，将Ｃ肽基因替换成含Ａｒｇ－Ａｒｇ－Ｇｌｙ－Ｓｅｒ－Ａｒｇ－Ｌｙｓ６个氨基酸小Ｃ肽基因。将这个小Ｃ肽岛素原类似物基因重组到具有Ｔａｃ启动子的质粒中，并与部分牛凝乳酶原基因融合，在Ｅ．ｃｏｌｉ中得到了高效表达。表达的ＢＣ＇Ａ融合蛋白占菌体总蛋白的１６％。表达产物以包含体形式存在，经ＣＮＢｒ裂解及磺酸，再经复性后，具有对胰岛素的放射免疫活性。