下颌骨牵引成骨过程中基因干预对牵引区转化生长因子β1表达的影响?****

 背景：局部基因治疗能促进牵引区新骨的生成,但关于基因治疗后对局部生长因子表达的影响目前尚不清楚。 目的：观察电穿孔介导的基因治疗对兔下颌骨牵引成骨过程中转化生长因子β1表达的影响。 方法：新西兰大白兔双侧下颌骨截骨后3 d开始下颌骨牵引,0.8 mm/d,连续牵引7 d后,随机分为5组,分别在牵引区注射2 μg(0.1 g/L)重组质粒pIRES-hVEGF165-hBMP2、pIRES-hBMP2、pIRES-hVEGF165、空质粒pIRES及相同剂量的生理盐水。之后施加电穿孔刺激。 结果与结论：免疫组织化学染色发现转化生长因子β1主要在细胞胞浆中表达,给药7 d时骨端骨细胞、编织骨痂骨细胞、骨痂表面成骨细胞呈转化生长因子β1染色阳性;14 d时新生成的编织骨痂骨细胞、骨痂表面成骨细胞、肉芽组织中的间质细胞、单核巨细胞、多核巨细胞转化生长因子β1染色阳性;28 d时转化生长因子β1阳性细胞明显减少。其中注射重组质粒pIRES-hVEGF165-hBMP2、pIRES-hBMP2、pIRES-hVEGF165后转化生长因子β1的表达明显多于注射空质粒pIRES及生理盐水(P < 0.05或P < 0.01)。说明基因治疗能促进转化生长因子β1的表达,促进牵引区细胞基质的形成和新骨生成。       

血管内皮细胞生长因子基因转染骨髓基质细胞的瞬时表达与稳定表达

目的探讨血管内皮细胞生长因子（VEGF）基因修饰的大鼠骨髓基质细胞（BMCS）的表达能力及表达活性。方法取6周龄SD大鼠BMCS传代培养后以3μl阳离子脂质体（Lipofectamine）：1μg pc DNA3．1-VEGF165的比例转染，通过RT—PCR技术、免疫组织化学S—P法检测转染细胞中外源性VEGF165基因的转录及瞬时表达和稳定表达，用血管内皮细胞（VEC）增殖试验测定转染细胞培养上清中VEGF的生物活性。结果VEGF基因转染的大鼠骨髓基质细胞可有效转录VEGF165，其分泌培养上清液中的表达产物可促进血管内皮细胞增殖，具有很强的生物活性。结论采用基因转染技术可将VEGF转染至BMCS中，并可有效表达具有生物活性的VEGF。     

血管内皮细胞生长因子mRNA在门脉高压脾脏中的表达

目的 了解血管内皮细胞生长因子mRNA在门脉高压脾脏中的表达。方法 逆转录-聚合酶链反应(RT-PCR)技术检测20例门脉高压小鼠脾脏VEGFmRNA表达。结果 有18例巨脾组织表达VEGF mRNA,表达率为90％;2例无表达。对照组为零。两组间有显著差异(p<0.01).结论 VEGF在门脉高压所致的脾肿大中起重要作用,通过诱导内皮细胞通透性和内皮细胞的增生,促进脾脏的肿大。     

可溶性血管内皮生长因子受体1真核克隆表达及其对血管内皮细胞增殖的影响

本研究旨在构建可溶性血管内皮生长因子(vascular endothelial growth factor,VEGF)受体1(soluble fmslike tyosine kinase-1,sFlt-1)的真核表达质粒pcDNA3.1-sFlt-1,并观察sFlt-1对血管内皮细胞增殖的影响。提取人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)总RNA,扩增Flt-1基因胞外1-3结构域,构建真核表达质粒pcDNA3.1-sFlt-1,测序鉴定基因序列。将重组质粒转染Lewis肺癌细胞,采用RT-PCR和SDS-PAGE检测sFlt-1在基因及蛋白水平的表达情况。MTT法检测sFlt-1对VEGF诱导的HUVECs生长的影响。结果显示:①插入片段序列正确;②sFlt-1在基因水平成功表达且转染后的Lewis肺癌细胞能分泌表达sFlt-1;③含sFlt-1的细胞上清液可明显抑制VEGF诱导的HUVECs增殖。本研究成功构建了真核表达质粒pcDNA3.1-sFlt-1,sFlt-1,在基因和蛋白水平均获得有效表达,且表达的蛋白可明显抑制由VEGF诱导...     

血管内皮细胞生长因子及临床应用策略

血管内皮细胞生长因子 (VEGF)是一种特异作用于血管内皮细胞的多功能细胞因子 ,它能引起血管通透性增加 ,引起细胞外基质成分改变 ,诱导血管形成。在炎症、创伤愈合、心脏缺血、动脉粥样硬化、糖尿病性视网膜病变及肿瘤形成等与血管生成和病变有关的诸多病理过程中起重要作用。     

血管内皮细胞生长因子与实体肿瘤

血管内皮细胞生长因子（ＶＥＧＦ）具有选择性促血管内皮细胞生长和增强血管渗透性的作用，在多种实体肿瘤中有高水平合成，并与肿瘤的分级和转移相关，ＶＥＧＦ受体主要表达于血管内皮细胞，通过干预ＶＥＧＦ及其受体抑制肿瘤血管形成是阻遏肿瘤生长和转移的重要策略     

血管内皮细胞生长因子及其受体的分子生物学研究进展

血管内皮细胞生长因子(vascular endothelial cell growth factor,VEGF)是最直接的血管内皮细胞促分裂素,它通过其受体(VEGFR)介导其活性,不同VEGF剪接体与不同类型的VEGFR结合,通过胞内信号传导,发挥不同的生物学效应。本文总结了VEGF及其受体的结构、特点、分类、分子生物学特征、二者的连结及由VEGF诱导的胞内信号传导作用,并简单讨论了它们可能的应用前景。     

血管内皮细胞生长因子受体KDR胞外配基结合区单抗对内皮细胞增殖及血管形成的抑制

目的　研制能封闭血管内皮生长因子 (VEGF)受体KDR的单克隆抗体 (McAb) ,探讨其抑制VEGF诱导的体内外活性。方法　以原核表达的KDRⅠ～Ⅳ区融合蛋白免疫BALB/c小鼠 ,用杂交瘤技术制备抗KDRⅠ～Ⅳ的McAb ,并用免疫双扩、ELISA和Western免疫印迹鉴定其亚类和抗原结合特异性 ,用VEGF刺激内皮细胞增殖及鸡胚血管形成实验检测该抗体的中和活性。结果　通过筛选和鉴定获得了 2株KDRⅠ～Ⅳ区McAbs(3G9、1E4) ,其分泌抗体亚类分别为IgG1和IgM。 2株McAb均能明显抑制VEGF诱导的内皮细胞 (EC)增殖 ,经纯化的McAb 3G9能明显抑制经VEGF刺激的鸡胚血管形成。结论　KDRⅠ～Ⅳ区McAb可能通过封闭内源性KDR而抑制VEGF活性 ,McAb 3G9具有潜在治疗肿瘤的应用前景。     

血管内皮细胞生长因子与大肠癌治疗

血管内皮细胞生长因子（VEGF）在大肠癌组织中高表达，与肿瘤血管生成，肿瘤细胞增殖、转移，肿瘤预后关系密切。目前各种针对VEGF及其受体开展的抑制肿瘤血管生成的药物研究广泛开展，有的已经批准上市，用于大肠癌的临床治疗，为大肠癌的治疗开辟了新的途径。     

腹腔液中巨噬细胞产生血管内皮细胞生长因子及其受体的表达

目的 :探讨腹腔环境在子宫内膜异位症发病机制中的作用 ,以及腹腔液血管内皮细胞生长因子 (VEGF)的来源和调节。方法 :将 14例子宫内膜异位症患者和 10例正常妇女腹腔液中的巨噬细胞体外培养 ,并在培养的正常妇女腹腔液巨噬细胞中加入 17 β雌二醇、孕酮和脂多糖 (LPS)共培养。用ELISA法检测培养的巨噬细胞上清液中VEGF的水平。同时将二者无血清培养的上清液 ,加入到内皮细胞中培养 ,用MTT比色法检测其对内皮细胞增殖活性的影响。用免疫组化法检测两者的巨噬细胞表达受体flt 1和flt 4的水平。结果 :子宫内膜异位症患者的巨噬细胞培养上清液中 ,VEGF的浓度明显高于正常对照组 (P <0 .0 5 ) ,且无周期性变化 (P >0 .0 5 )。子宫内膜异位症患者的巨噬细胞无血清培养上清液 ,在体外能显著提高内皮细胞的增殖活性 (P <0 .0 5 )。雌激素和孕激素能显著增加体外巨噬细胞分泌VEGF的水平 (P <0 .0 5 ) ,与LPS组相比较差异显著 (P <0 .0 5 ) ,即激素的调节作用大于LPS的作用。但雌激素和孕激素调节巨噬细胞分泌VEGF的程度无明显差别 (P >0 .0 5 )。LPS激活的巨噬细胞能增加雌激素、孕激素对巨噬细胞分泌VEGF的调节作用 (P <0 .0 5 )。巨噬细胞能表达受体flt 1和flt 4 ,无周期性变化 (P >0 .0 5 )。结论 :子宫内膜     

携带人血管内皮生长因子165慢病毒表达载体的构建与表达*

背景：已有研究证实血管内皮生长因子在正常肝脏肝部分切除后余肝的再生过程中发挥着重要的作用,但关于其对肝硬化肝脏是否也有相同作用国内外鲜有报道。 目的：构建携带人血管内皮生长因子165基因的慢病毒载体,体外转染BLR 3A大鼠肝细胞并观察该细胞中人血管内皮生长因子165基因的表达。 方法：采用DNA重组技术将人血管内皮生长因子165基因克隆入pLenti6/V5-D-TOPO慢病毒表达载体中,筛选出阳性克隆与慢病毒包装系统ViraPowerTM Packaging Mix共转染293T细胞产生病毒颗粒,通过实时定量-聚合酶链反应法测定病毒滴度;携带人血管内皮生长因子165基因的慢病毒载体体外转染BLR 3A大鼠肝细胞72 h后,利用反转录-聚合酶链反应及Western blot法检测细胞中人VEGF165 mRNA及蛋白的表达。 结果与结论：成功构建了表达人VEGF165基因的慢病毒载体pLenti6/V5-D-TOPO-VEGF165,测得病毒滴度为1.18×     107 VP/mL。重组慢病毒载体转染BLR 3A大鼠肝细胞72 h后,荧光蛋白表达率超过80%,反转录-聚合酶链反应及Western blot法测得转染组人血管内皮生长因子165 mRNA及血管内皮生长因子165蛋白表达阳性。提示构建的携带人血管内皮生长因子165的慢病毒载体可有效转染BLR 3A大鼠肝细胞,并促使该细胞表达人血管内皮生长因子165 mRNA及蛋白。 关键词：人血管内皮生长因子165;慢病毒载体;BLR 3A大鼠肝细胞;转染;基因治疗 doi:10.3969/j.issn.1673-8225.2012.11.007     

TGF-β对缺氧环境下HepG2细胞表达VEGF的影响

目的探讨转化生长因子β1(TGF-β1)和低氧(CoCl2)对肝癌细胞血管内皮生长因子(VEGF)表达的影响。方法体外培养肝癌细胞系HepG2细胞,实验分为对照组、不同浓度TGF-β1和CoCl2的分别处理组、TGF-β1(100 pmol/L) CoCl2(200 μmol/L)组;用免疫细胞化学法检测VEGF蛋白表达;半定量RT-PCR技术检测细胞中VEGFmRNA相对表达量。结果各处理组VEGF蛋白表达均为阳性;TGF-β1或CoCl2组中VEGF mRNA相对表达量明显高于对照组(P<0.01),并且呈浓度依赖性;TGF-β1 CoCl2组中VEGFmRNA表达高于单因素刺激组(P<0.05)。结论TGF-β1能够刺激HepG2细胞表达VEGF,并且可以协同CoCl2增加VEGF的表达。     

Effects of vascular endothelial growth factor expression on pathological characteristics and prognosis of osteosarcoma

Vascular endothelial growth factor (VEGF) has been linked with tumor invasion and metastasis. However, the role of VEGF expression in osteosarcoma remains controversial. By searching the PubMed, Embase, and Google Scholar databases, we conducted a meta-analysis to evaluate the pathological and prognostic significance of VEGF in osteosarcoma. Studies were pooled, and the odds ratio (OR) and its corresponding 95 % confidence interval (CI) were calculated. Nine relevant articles were included in this meta-analysis study. We performed pooled analysis with available data on the association between VEGF expression and age, gender, tumor stages IIB–III versus I–IIA, tumor recurrence, response to chemotherapy, and tumor metastasis. Our results revealed that VEGF expression might be closely associated with metastasis of osteosarcoma (OR 4.74, 95 % CI 2.53–8.87, P < 0.001). Furthermore, our findings also demonstrated that patients with grade IIB–III osteosarcoma showed a higher frequency of VEGF expression than those with grade I–IIA osteosarcoma (OR 5.33, 95 % CI 2.03–13.98, P = 0.001). We failed to find the association between VEGF expression and age (OR 0.82, 95 % CI 0.44–1.53, P = 0.539), gender (OR 1.33, 95 % CI 0.52–3.42, P = 0.553), tumor recurrence (OR 1.47, 95 % CI 0.56–3.86, P = 0.429), and response to chemotherapy (OR 1.26, 95 % CI 0.14–11.72, P = 0.839). In conclusion, VEGF is related to the grade and metastasis of osteosarcoma. It may play a significant role in clinical guidelines for the treatment and prognostic evaluation.     

Hypoxia and cobalt stimulate vascular endothelial growth factor receptor gene expression in rats

 This study aimed to examine the influence of acute tissue hypo-oxygenation on the expression of the vascular endothelial growth factor (VEGF) receptor genes. To this end male Sprague-Dawley rats were exposed to different hypoxic conditions such as 10% or 8% oxygen, 0.1% carbon monoxide and cobalt chloride (60 mg/kg) for 6 h and the abundance of flt-1, flt-4 and flk-1 mRNA in lungs and livers was determined by RNase protection assay. The relative proportions of flt-1, flt-4 and flk-1 were 10 : 2.5 : 1 and 10 : 10 : 2 in normoxic lungs and livers, respectively. It was found that 8% but not 10% oxygen increased flt-1 mRNA two- to threefold in both organs, whilst flt-4 and flk-1 mRNA were not changed by acute inspiratory hypoxia. Carbon monoxide inhalation also increased flt-1 mRNA but not flt-4 or flk-1 mRNA in both organs. Subcutaneous cobalt administration increased flt-1 mRNA in the livers only, whilst flt-4 and flk-1 mRNA remained unchanged. These findings show that acute tissue hypo-oxygenation is a rather selective stimulus for flt-1 gene expression. The efficiency of the different manoeuvres applied to stimulate flt-1 gene expression is rather similar to the stimulation of erythropoietin gene expression. It is not unreasonable to assume, therefore, that the oxygen-dependent regulation of both genes at the cellular level has significant similarities.     

Syk基因对乳腺癌血管内皮生长因子-C表达的调控作用

目的 研究Syk基因对乳腺癌血管内皮生长因子(VEGF)-C表达的调控机制.方法 采用免疫组织化学(EnVision法)检测乳腺癌组织中Syk、NFκB与VEGF-C的蛋白表达情况,分析三者的相关性及与淋巴转移的关系.转染pcDNA3.1(-)-Syk至乳腺癌MDA-MB-231细胞,检测对VEGF-C和NFκB表达的影响.结果 在淋巴转移组中,Syk蛋白阳性率低于非淋巴转移组,VEGF-C与NFκB阳性率在淋巴转移组高于非淋巴转移组.Syk蛋白表达与NFκB(r=-0.448,P=0.002)、VEGF-C(r=-0.620,P=0.000)蛋白表达呈负相关,VEGF-C与NFκB呈正相关(r =0.310,P=0.036).转染pcDNA3.1(-)-Syk重组质粒的乳腺癌细胞中VEGF-C的mRNA及蛋白表达水平均低于空白对照组,细胞核NFκB蛋白表达低于空白对照组(均P＜0.05).结论 Syk基因与乳腺癌转移相关,可能是通过抑制NFκB的活性而下调VEGF-C的表达,从而抑制乳腺癌的淋巴转移.     

High flow drives vascular endothelial cell proliferation during flow-induced arterial remodeling associated with the expression of vascular endothelial growth factor

Endothelial cell activation and proliferation are the essential steps in flow-induced arterial remodeling. We investigated endothelial cell turnover in the early stages of high-flow in the rabbit common carotid arteries using an arteriovenous fistula (AVF) model by kinetic investigation of cell proliferation and cell molecular analysis. BrdU was administrated to label endothelial cells (ECs) in DNA synthetic phase (S-phase) of the cell mitotic cycle. Pulse labeling revealed that ECs entered S-phase at 1.5 days of AVF (0.93 +/- 0.19%). Endothelial cell labeling index (EC-LI) peaked at 2 days of AVF (8.90 +/- 0.87%) with a high index of endothelial cell mitosis (EC-MI, 1.67 +/- 0.47%). Endothelial cell density increased remarkably at 3 days of AVF with a significant decrease in EC-LI (54%) and EC-MI (60%). Study of kinetics of EC proliferation revealed that endothelial cells took 16-24 h to finish one cycle of cell mitosis. Tracking investigation of pulse BrdU-labeled endothelial cells at 1.5 days showed that more than 66% of endothelial cells were BrdU-labeled 1.5 days after labeling. VEGF, integrin alphanubeta3, PECAM-1, and VE-cadherin were upregulated significantly preceding endothelial cell proliferation and kept at high levels during endothelial cell proliferation. These data suggest that endothelial cell proliferation is the initial step in flow-induced arterial remodeling. Hemodynamic forces may drive endothelial cell downstream migration. Expression of VEGF and cell junction molecules contribute to flow-induced arterial remodeling.     

Effect of hypericin-mediated photodynamic therapy on the expression of vascular endothelial growth factor in human nasopharyngeal carcinoma

Photodynamic therapy (PDT) is currently being used as an alternative treatment modality for various types of cancers. PDT involves the selective uptake and retention of a photosensitizer in the tumor followed by light irradiation of an appropriate wavelength to cause the destruction of tumor cells by the formation of cytotoxic reactive oxygen species. The photosensitizer, hypericin, has shown great potential due to its light-dependent tumor destructive properties. However, as hypericin-mediated PDT primarily targets tumor vasculature, it induces certain pro-angiogenic factors such as vascular endothelial growth factor (VEGF) in the tumor tissue as a result of hypoxia. This study examines the role of hypericin-mediated photodynamic therapy in stimulating the expression of key angiogenesis growth factor VEGF in order to elucidate the process of tumor angiogenesis in nasopharyngeal carcinoma xenografts. We also investigated the effect of angiogenesis inhibitor celebrex on human VEGF levels when combined with hypericin-PDT. These studies were conducted on an in vivo human nasopharyngeal xenograft model. VEGF was measured in the control and hypericin-PDT treated tumors. VEGF levels were found to be higher when the tumors were treated at a 1-h drug-light interval compared to a 6-h interval, due to extensive vascular damage. At 72 h post hypericin-PDT, VEGF levels were upregulated indicating the initiation of regrowth in tumors. The use of angiogenesis inhibitor, celebrex, along with hypericin-PDT downregulated the human VEGF levels suggesting that angiogenesis inhibitors can be used to improve the outcome of hypericin-PDT in nasopharyngeal carcinomas.     

Divergent regulation of vascular endothelial growth factor and of erythropoietin gene expression in vivo

There is accumulating evidence from in vitro experiments that the gene expression of the vascular endothelial growth factor (VEGF) is, like that of the erythropoietin (EPO) gene, regulated by the oxygen tension and by divalent cations such as cobalt. Since the information about the regulation of VEGF gene expression in vivo is rather scarce, this study aimed to examine the influence of hypoxia and of cobalt on VEGF gene expression in different rat organs and to compare it with that on EPO gene expression. To this end male Sprague-Dawley rats were exposed to carbon monoxide (0.1% CO), hypoxia (8% O₂ ) or to cobalt chloride (12 and 60 mg/kg s.c.) for 6 h. mRNA levels for VEGF- 188, -164, and -120 amino acid isoforms in lungs, hearts, kidneys and livers were semiquantitated by RNase protection. For these organs we found a rank order of VEGF mRNA abundance of lung >> heart > kidney = liver. EPO mRNA levels were semiquantitated in kidneys and livers. Hypoxia, CO and cobalt increased EPO mRNA levels 60-fold, 140-fold and 5-fold, respectively, in the kidneys, and 11-fold, 11-fold and 3-fold, respectively, in the livers. None of these manoeuvres caused significant changes of VEGF mRNA in lung, heart or kidneys. Only in the livers did hypoxia lead to a significant (50%) increase of VEGF mRNA. These findings suggest that, in contrast to the in vitro situation, the expression of the VEGF gene in normal rat tissues is rather insensitive to hypoxia. In consequence, the in vivo regulation of the VEGF and the EPO genes appear to differ substantially, suggesting that the regulation of the VEGF and EPO genes may not follow the same essential mechanisms in vivo. Received: 31 July 1995/Received after revision: 20 November 1995/Accepted: 27 November 1995