缺氧反应元件启动子表达反义血管内皮生长因子(VEGF)165 cDNA靶向性抑制骨肉瘤细胞VEGF表达

目的以缺氧诱导因子-1/缺氧反应元件(HIF-1/HRE)基因调节系统为载体,构建含HRE启动子的人反义血管内皮生长因子(VEGF)165 cDNA真核表达载体,探讨其对缺氧条件下骨肉瘤细胞VEGF表达的靶向性抑制作用.方法采用聚合酶链反应(PCR)和DNA重组技术构建由HRE启动子驱动的含荧光素酶(Luc)报告基因和人反义VEGF165 cDNA全长的真核表达质粒,将其转染骨肉瘤细胞系MG63,用液闪计数仪测定缺氧对报告基因表达的调节,酶链免疫吸附试验(ELISA)法检测缺氧条件下细胞VEGF表达的变化.结果成功构建了由HRE启动子驱动的含Luc和反义VEGF165 cDNA全长的真核表达质粒pBI-HRE-Luc-AsVEGF165,该质粒转染骨肉瘤细胞系,缺氧条件下可使报告基因在肿瘤细胞中的表达提高3.5×102倍,使肿瘤细胞VEGF分泌下降45%.结论利用肿瘤缺氧,HRE启动子能够实现反义VEGF165的高效表达,为开展靶向性抗肿瘤血管生成治疗提供了实验依据.     

Fascin在骨肉瘤中的表达及意义

目的探讨fascin蛋白在骨肉瘤中的表达变化与临床病理指标及生存时间的关系。方法应用免疫组织化学方法和Western blot方法检测45例手术切除骨肉瘤标本fascin蛋白的表达,将所得fascin蛋白表达的光密度值数据与临床病理指标及生存时间进行统计学分析。结果肿瘤边缘的非瘤骨组织中无fascin蛋白表达,45例骨肉瘤标本中有24例fascin蛋白为中高表达,这种阳性表达与外科分期、化疗反应性呈正相关(P<0.05)。在单因素及多因素分析中,蛋白fascin的高表达的骨肉瘤患者总生存期及无病生存期明显降低(P<0.05)。结论fascin基因可能是骨肉瘤的一个潜在治疗靶点。     

TRAIL及NF-kB在骨肉瘤中的表达及其意义

本文探讨TRAIL及NFkB在骨肉瘤中的表达及其与细胞增殖调控的关系。对16例非化疗骨肉瘤,5例骨巨细胞瘤和6例软骨肉瘤石蜡切片,应用流式细胞术(FCM)定量检测TRAIL及NFkB的表达。结果表明TRAIL和NFkB在骨肉瘤各期间的表达显著高于骨巨细胞瘤和软骨肉瘤(P<0.01);TRAIL和NFkB在骨肉瘤不同分化组间表达无明显差异(P>0.05);TRAIL和NFkB在骨巨细胞瘤和软骨肉瘤间表达无明显差异(P>0.05)。结论是TRAIL在骨肉瘤中高表达,但可能受NFkB高表达的调控,不能诱导细胞凋亡;在骨肉瘤的细胞增殖调控中,NFkB可能通过调控TRAIL诱导的凋亡途径进而抑制细胞凋亡的发生。     

骨肉瘤组织survivin与 VEGF的表达及相关性

凋亡抑制蛋白（inhibitor of apoptosis protein,IAP）是抑制细胞凋亡的重要成分,survivin是最近发现的一种IAP家族成员,其在成人正常组织中不表达而选择性表达于恶性肿瘤组织的特性,使抗细胞凋亡与肿瘤发生的关系更为接近。血管内皮细胞生长因子（vascular endothelium growth factor,VEGF）是已知肿瘤血管生成中最重要的诱发因子,     

普通骨肉瘤中CD44、纤连蛋白的表达及意义

ＣＤ44是一类特殊的黏附分子 ,能参与细胞与细胞之间以及细胞与细胞外基质之间的相互作用。纤连蛋白(ｆｉｂｒｏｎｅｃｔｉｎ ,ＦＮ)是细胞外基质的重要组分 ,参与细胞分化、迁移以及肿瘤浸润等过程。本文将探讨普通骨肉瘤中标准型ＣＤ44 (ＣＤ44ｓ)和ＦＮ的表达及意义。一、材料与方法1 临床资料 :收集本教研室 1995年 7月～ 2 0 0 1年 10月骨肉瘤手术标本 6 2例 ,发病年龄 9～ 49岁 ,中位年龄 17岁 ;术后复发时间 5个月～ 3年 ;标本经常规 4%中性甲醛固定 ,石蜡包埋 ,重染ＨＥ复查。共收集初发性普通骨肉瘤 5 1例(其中成骨为主型 35例 ,成…     

骨肉瘤的细胞遗传学及分子遗传学研究进展

本文介绍了骨肉瘤病人的细胞遗传学的核型变化情况，以及使用Ｓｏｕｔｈｅｒｎ，Ｎｏｒｔｈｅｒｎ印迹、斑点杂交及原位杂交、ＲＦＬＰ、ＰＣＲ、ＳＳＣＰ等技术研究骨肉瘤的分子遗传学。     

湍流流动对血管内皮细胞黏附分子表达的影响

本研究考察了ECs在不同流场中的血管细胞黏附分子(VCAM-1)和胞间黏附分子(ICAM—1)表达，揭示了湍流流体力学信号影响黏附分子表达和ECs功能状态。构建了层流和湍流的离体流室模型，运用共聚焦显微镜，从蛋白水平考察培养的人脐静脉ECs中VCAM-1和ICAM-1在不同流室中的表达。发现层流中VCAM-1表达显著增强，而ICAM-1表达只一过性增加，随即回落。湍流中VCAM-1表达下降，而ICAM-1表达持续缓慢上升。由此表明：湍流流场对血管ECs黏附特性的影响不同于层流，而且不同的黏附分子对流动具有不同的响应性；湍流是引起ECs形态结构和功能行为的病理学改变的重要原因。     

Ezrin在骨肉瘤中的表达及临床意义

目的探讨人体骨肉瘤组织中的Ezrin mRNA和蛋白的表达及其与骨肉瘤临床病理特征的关系。方法收集42例骨肉瘤和15例良性骨肿瘤标本,应用免疫组织化学技术检测Ezrin的蛋白表达,应用RT-PCR检测Ezrin mRNA的表达情况,并与临床病理特征进行相关性分析。结果 Ezrin蛋白及mRNA在骨肉瘤中的阳性表达显著高于其在良性骨肿瘤中的阳性表达（P〈0.05）;Ezrin mRNA表达的阳性率和骨肉瘤分化程度密切相关,而与患者性别、年龄及肿瘤部位无关。结论 Ezrin可能参与了骨肉瘤的发生、发展和转移,Ezrin的表达情况可作为评估骨肉瘤生物学行为的一项指标。     

免疫球蛋白超家族黏附分子在淋巴管和不同血管内皮细胞的表达

目的比较免疫球蛋白超家族黏附分子在淋巴管、大血管和微血管内皮细胞的表达特点,探讨免疫球蛋白超家族黏附分子在淋巴管内皮细胞表达的意义。方法从狗的胸导管、颈总动脉、颈内静脉、肺微血管分离内皮细胞,利用免疫荧光标记法检测PECAM-1、ICAM-1、ICAM-3、VCAM-1和CD44在各种内皮细胞的表达,在荧光显微镜和激光共聚焦扫描显微镜下观察,并用图像分析仪分析表达强度。结果动脉、静脉和肺微血管内皮细胞表达PECAM—1、ICAM—1、ICAM-3、VCAM—1和CD44。其中,ICAM-1和ICAM-3的表达较弱。VCAM—1在动脉和肺微血管内皮细胞的表达比静脉强。淋巴管内皮细胞表达PECAM—1、ICAM—1、ICAM-3和CD44,未观察到VCAM—1的表达。ICAM-3和CD44的表达比血管内皮细胞强。结论与动脉、静脉和微血管内皮细胞比较,淋巴管内皮细胞不表达VCAM—1,而ICAM-3和CD44表达较强,这有助于解释淋巴细胞和肿瘤细胞与淋巴管内皮的黏附以及淋巴管新生的机制。     

血小板内皮细胞黏附分子的剪接体在胚胎干细胞血管形成过程中的表达

目的探讨血小板内皮细胞黏附分子(PECAM)1的剪接体在胚胎干细胞分化过程中的表达规律。方法体外培养胚胎干细胞,在细胞因子的作用下形成胚胎样小体(EB);然后将EB种植到胶原中,在细胞因子的作用下诱导出芽性血管新生。分别利用免疫组织化学、流式细胞术、逆转录聚合酶链反应等方法检测胚胎干细胞及其分化过程中PECAM1、Oct4及胚胎阶段特异性抗原(SSEA)1的表达。应用克隆分析的方法检测不同的PECAM1剪接体在EB形成和出芽性血管新生过程中的表达分布。     

血管内皮生长因子(VEGF)对培养内皮细胞VEGF受体表达的影响

目的 :为探讨VEGF对培养内皮细胞 (EC)VEGF受体表达的影响。方法 :将培养的人脐静脉内皮细胞 (HUVEC)随机分为 4组 :( 1)正常对照组 ;( 2 )低氧培养组 ;( 3)VEGF 10ng/ml组 ;( 4)低氧 +VEGF10ng/ml组。HUVEC低氧培养参照Kuwara等介绍的方法并加以改进。HUVECVEGF受体的检测采用免疫组织化学方法。结果 :采用简易低氧培养法 ,48h内培养液氧分压维持在 5 8mmHg ;与对照组相比 ,低氧培养组、VEGF组和低氧 +VEGF组HUVECVEGF受体Flk 1/KDR阳性细胞数增多 ,强度增加 ;但未检测到VEGF受体Flt 1表达。结论 :低氧可使HUVEC表面的VEGF受体Flk 1/KDR表达增加 ,VEGF同源上调其受体Flk 1/KDR ,低氧和VEGF在调节VEGF受体Flk 1/KDR方面有协调作用。     

Vascular Endothelial Growth Factor (VEGF) in Autoimmune Diseases

Vascular endothelial growth factor (VEGF) is a potent stimulating factor for angiogenesis and vascular permeability. There are eight isoforms with different and sometimes overlapping functions. The mechanisms of action are under investigation with emerging insights into overlapping pathways and cross-talk between other receptors such as the neuropilins, which were not previously associated to angiogenesis. VEGF has important physiological actions on embryonic development, healing, and menstrual cycle. It also has a great role in pathological conditions that are associated to autoimmune diseases. There is considerable evidence in various autoimmune diseases such as in systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis of an interrelationship between the VEGF system and theses disorders. Serum levels of VEGF correlate with disease activity in a large number of autoimmune diseases and fall with the use of standard therapy. We raised the possible future therapeutic strategies in autoimmune diseases with the anti-VEGF or anti-VEGFR (receptor). So far, this therapy has been used in cancer and macular ocular degeneration in diabetes. This review outlines the evidence for VEGF participation in various autoimmune diseases and proposes lines for future research in this field.     

Immunohistochemical Expression of Vascular Endothelial Growth Factor in Canine Mammary Tumours

The histopathological and clinical aspects of canine mammary tumours (CMTs) have been widely studied, but the variation in the biological behaviour of these neoplasms hampers the identification of prognostic factors. Sustained angiogenesis has been suggested to be one of the most important factors underlying tumour growth and invasion. This process involves the action of several growth factors including vascular endothelial growth factor (VEGF). The present study characterizes the relationship between immunohistochemical expression of VEGF and gross (e.g. size and tissue fixation) and microscopical (e.g. type, growth, necrosis, lymphoid infiltration, lymph node metastasis, histological grade and proliferation index) features of CMTs. Forty-eight benign and 64 malignant CMTs were evaluated. Statistical analysis failed to show a significant relationship between VEGF expression and the pathological features, suggesting that VEGF expression occurs in both benign and malignant tumours and is independent of histological type, proliferation, tissue invasion or local metastatic capacity.     

人脑胶质瘤VEGF的表达和微血管检测及意义

目的探讨人脑胶质瘤中血管内皮细胞生长因子(VEGF)的表达与微血管数量(MVQ)和肿瘤良恶性程度的关系. 方法用免疫组织化学染色方法检测手术切除石蜡包埋的47例人脑胶质瘤(Ⅰ～Ⅱ级22例,Ⅲ级15例,Ⅳ级10 例)组织中的VEGF蛋白表达情况和MVQ数量,借助显微镜观察其阳性细胞数和阳性血管数; 另外,分别取脑外伤后开颅内减压术中切除的正常脑组织10例作为对照组. 结果①正常脑组织未检测到VEGF蛋白的表达,而MVQ数量少 ,表达强度弱;②各级别胶质瘤组织中均有VEGF和MVQ表达,主要表达于肿瘤细胞的胞浆和血管内皮细胞胞膜中;③3组胶质细胞瘤中VEGF的表达阳性率分别为22.2%、86.7%、90%、Ⅰ～Ⅱ级组与其它两组间差异有显著性意义(p<0.05;p<0.01),VEGF表达阳性组的MVQ平均值(46.8±12.4)明显高于阴性组MVQ值 (22.4±11.5)(p<0.01);④VEGF的表达与MVQ相关(r=0.75, p <0.001). 结论①VEGF在脑胶质瘤血管生成中起重要作用 ,能促进胶质瘤血管的形成;②VEGF和MVQ与胶质瘤良恶性程度明显相关,可作为脑胶质瘤病理诊断的补充指标;③VEGF可作为治疗脑胶质瘤的靶向,为胶质瘤的基因治疗提供指导方向.     

Expression of Vascular Endothelial Growth Factor Isoforms in the Japanese Quail Embryo

Vascular endothelial growth factor (VEGF) is required for vascular development. In the quail, four VEGF isoforms formed by alternative splicing, are translated into proteins with 122, 146, 166, and 190 amino acids. VEGF isoforms differ biochemically, with variable affinities for heparan sulfate proteoglycans, the extracellular matrix and VEGF receptors. There are few data on the functional significance of VEGF isoforms. RT-PCR was used to examine isoform expression during quail vascular development. Our results suggest that all quail isoforms are expressed during establishment of the vascular pattern in whole embryos and extraembryonic tissues at apparently equal levels. No isoform-specific expression patterns were detected in isolated endoderm or between embryo halves.     

脂质体介导的VEGF_(165)基因转染对内皮细胞生长的作用

构建血管内皮细胞生长因子基因VEGF165真核表达载体pcDNA3 VEGF165,以阳离子脂质体介导的基因转染技术 ,将基因转入原代培养的人脐静脉内皮细胞中。结果表明 ,基因转染 1h后细胞内就有VEGFDNA存在 ,VEGFmRNA水平显著上升 ;基因转染 2d后培养液上清VEGF蛋白表达显著上升 (135 5 12± 6 2 34)pg/ml和 (19 2 7± 2 96 )pg/ml,P <0 0 1。转染VEGF165基因 2d的内皮细胞再经程序降温冷冻保存复苏后 ,其存活率显著高于对照组 (pcDNA3 组 ) (90 13%± 2 84 %和81 5 2 %± 2 15 % ,P <0 0 5 ) ,凋亡率显著低于对照组 (7 15 %± 0 4 2 %和 17 6 1%± 1 5 6 % ,P <0 0 5 )。MTT法显示转染VEGF165基因能促进内皮细胞VEGF蛋白的表达 ,促进细胞增殖 ,抑制细胞凋亡。在治疗心脏及下肢动脉缺血性疾病中 ,VEGF165基因治疗可能具有重要的意义。     

Isoflavone regulates Vascular Endothelial Growth Factor Expression in urinary tract of castrated rats

Objective

The purpose of this study was to investigate Vascular Endothelial Growth Factor Expression (VEGF) gene regulation by isoflavone in urinary tract tissues of castrated adult rats.

Design

Forty-five adult rats, 90 days old, weighting 200 g were used, receiving a soy-free ration. The animals were castrated for drug administration for 30 days (125 μg genisteine/g body weight/day) and sacrificed, divided into three groups: Group I—control; Group II—started isoflavone administration on the 5th day after castration; Group III—started isoflavone administration on the 28th day after castration. RNA was isolated from each bladder and urethra. Determination of VEGF gene regulated by isoflavone was obtained using a semiquantitative RT-PCR and immunohistochemistry of total RNA isolated from bladder and urethra.

Results

Our results demonstrate that isoflavone was able to upregulate mRNA level of the VEGF gene in the lower urinary tract of rats in Group II, where isoflavone administration was started at an early phase of estrogen deprivation, while in Group III, where isoflavone administration was started in the late phase of hypoestrogenism, did not show alteration of bladder and urethra VEGF gene expression, compared to placebo, maintaining the same level of the castrated rats without treatment.

Conclusions

The data indicate that VEGF expression in rats is also regulated by isoflavone in early phase of hypoestrogenism.     

Covalently-Immobilized Vascular Endothelial Growth Factor Promotes Endothelial Cell Tubulogenesis in Poly(ethylene glycol) Diacrylate Hydrogels

The development and use of functional tissue-engineered products is currently limited by the challenge of incorporating microvasculature. To this end, we have investigated strategies to facilitate vascularization in scaffold materials, in this case poly(ethylene glycol) (PEG) hydrogels. These hydrogels are hydrophilic and resist protein adsorption and subsequent non-specific cell adhesion, but can be modified to contain cell-adhesive ligands and growth factors to support cell and tissue function. Additionally, the hydrogel matrix can include proteolytically degradable peptide sequences in the backbone of the structure to allow cells to control scaffold biodegradation, allowing three-dimensional migration. Vascular endothelial growth factor (VEGF), a potent angiogenic signal, and the cell-adhesive peptide RGDS were each covalently attached to PEG monoacrylate linkers. PEGylated RGDS and VEGF were then covalently immobilized in PEG-diacrylate (PEGDA) hydrogels in 2D and 3D. Immobilized VEGF increased endothelial cell tubulogenesis on the surface of non-degradable PEGDA hydrogels 4-fold compared to controls without the growth factor. Endothelial cell behavior in 3D collagenase-degradable hydrogels modified with RGDS and VEGF was observed using time-lapse confocal microscopy. Bulk immobilization of VEGF in 3D collagenase-degradable RGDS-modified hydrogels increased endothelial cell motility 14-fold and cell–cell connections 3-fold. Covalent incorporation of PEGylated VEGF in PEG hydrogels can be a useful tool to promote endothelial cell migration, cell–cell contact formation and tubulogenesis in an effort to produce vascularized tissue-engineered constructs.     

血管内皮生长因子分子多样性及其与肿瘤的关系

血管内皮生长因子是肿瘤发生发展过程中起重要作用的一类糖蛋白,其在基因水平存在较多的SNP位点以及在RNA剪切水平上存在选择性剪切.研究表明,这些变异与各种肿瘤的发生、发展、恶性程度有着密切的关系.