缺氧诱导因子1α小干扰RNA对骨髓间充质干细胞缺氧诱导因子1α、基质细胞衍生因子1α和血管内皮生长因子基因表达的影响*

背景：缺氧可以影响骨髓间充质干细胞分泌细胞因子,其机制可能是通过缺氧诱导因子1α起作用的。 目的：观察缺氧诱导因子1α RNA干扰对骨髓间充质干细胞缺氧诱导因子1α、基质细胞衍生因子1α和血管内皮生长因子基因表达的影响。 方法：贴壁法培养骨髓间充质干细胞,取第3~5代细胞用于实验,倒置显微镜下观察细胞形态,并用流式细胞仪检测其表面标志物CD34、CD44、CD90表达。将骨髓间充质干细胞分四组：常氧对照组(无干预因素)、缺氧组(缺氧24 h)、脂质体对照组(转染空载脂质体后缺氧24 h)、RNA干扰组(转染脂质体介导的RNA干扰序列后缺氧24 h)。RT-PCR法检测骨髓间充质干细胞的缺氧诱导因子1α、基质细胞衍生因子1α和血管内皮生长因子 mRNA表达水平, ELISA检测骨髓间充质干细胞培养上清缺氧诱导因子1α、基质细胞衍生因子1α和血管内皮生长因子蛋白表达水平。 结果与结论：同常氧对照组比较,缺氧组缺氧诱导因子1α、基质细胞衍生因子1 α和血管内皮生长因子基因及蛋白表达增高(P < 0.05);同脂质体对照组比较,RNA干扰组缺氧诱导因子1α、基质细胞衍生因子1α和血管内皮生长因子基因及蛋白表达降低(P < 0.05)。证实了缺氧条件可以使骨髓间充质干细胞血管内皮生长因子和基质细胞衍生因子1α的表达增加,抑制缺氧诱导因子1α的表达可以使血管内皮生长因子和基质细胞衍生因子1α的表达减少,缺氧诱导因子1α很可能是干细胞移植细胞因子分泌的调控因素。     

糖尿病脑梗死大鼠血管内皮生长因子及其受体表达水平的变化

目的 研究糖尿病脑梗死(DMCI)早期半暗带等值区血管内皮生长因子(VEGF)及其受体(FLT-1,FLK-1)表达水平的变化.方法 建立DMCI大鼠模型,在脑缺血后1 h、3 h、6 h、12 h、24 h,采用原位杂交方法测定大鼠大脑缺血半暗带等值区VEGF、FLT-1、FLK-1 mRNA的表达水平;并与无糖尿病的脑梗死(NDMCI)大鼠比较.结果 DMCI组在脑缺血后各时间点脑缺血半暗带等值区VEGF mRNA的表达水平均显著低于NDMCI组(P＜0.05～0.01),FLK-1 mRNA表达水平在脑缺血后12 h显著低于NDMCI组(P＜0.01),FLT-1 mRNA表达水平在脑缺血后各时间点(除6 h)与NDMCI组差异无统计学意义.结论 DMCI早期VEGF和FLK-1 mRNA表达水平降低,这可能是DMCI血管、神经病变重的原因之一.     

基质细胞源性生长因子1α和血管内皮生长因子在退变椎间盘组织中的表达及意义

背景：在正常情况下成人椎间盘组织中含有很少量的小血管，但在突出的椎间盘中却出现了明显的血管化，基质细胞源性生长因子1α在人类出生后成人组织血管发生的启发过程中起到了非常重要的作用，但在椎间盘中的表达少见报道。 目的：检测基质细胞源性生长因子1α和血管内皮生长因子在腰椎间盘中的表达,进而评估两者在椎间盘的血管化中的作用。 设计、时间及地点：对比观察实验，于2006-05/2007-01在中国医科大学组织工程实验室完成。 材料：48份椎间盘突出标本及14份非椎间盘突出标本。椎间盘突出标本分突出型、脱出型、游离型。 方法:采用SABC免疫组织化学技术对实验组48份腰椎间盘突出标本及对照组14份非腰椎间盘突出标本进行抗SDF-1α和抗 VEGF单克隆抗体染色。 主要观察指标：腰椎间盘中基质细胞源性生长因子1α及血管内皮生长因子的表达。 结果:实验组椎间盘标本中基质细胞源性生长因子1α和血管内皮生长因子的表达均高于对照组标本，实验组标本中游离型及脱出型的基质细胞源性生长因子1α和血管内皮生长因子蛋白表达均比突出型高，游离型与脱出型之间的表达无差异。 结论: 腰椎间盘组织中基质细胞源性生长因子1α和血管内皮生长因子的表达可能与腰椎间盘血管新生有关。     

兔耳瘢痕成熟过程中血管内皮细胞生长因子与缺氧诱导因子1α的表达

背景：尽管近20年来在组织、细胞和分子各个水平上展开了瘢痕的研究,但瘢痕形成的完整机制尚未阐明,探索瘢痕的形成的分子机制,寻求理想的瘢痕防治方法是医学研究和实践中一项紧迫而艰巨的任务。 目的：观察兔耳瘢痕从形成到消退成熟过程中血管内皮细胞生长因子与缺氧诱导因子1α变化,探讨瘢痕形成的分子机制。 方法：制作兔耳瘢痕从形成到消退成熟的动物模型,收集兔耳术后14,30,60和90 d瘢痕和正常皮肤,应用苏木精-伊红染色方法观察兔耳术后各时间点瘢痕的组织形态学变化,免疫组织化学法方法检测术后各时间点血管内皮细胞生长因子、缺氧诱导因子1α在瘢痕组织中的表达。 结果与结论：术后14 d瘢痕组织中血管内皮细胞生长因子和缺氧诱导因子1α表达较其他时间点和正常组织均升高,而随着时间的延长,瘢痕的萎缩成熟,两者的表达水平逐渐降低,并在术后90 d达到正常组织表达水平。结果表明缺氧诱导因子1α与血管内皮细胞生长因子在瘢痕组织内氧分压的变化过程中起到一定的调节作用。     

缺氧诱导因子-1α和血管内皮生长因子与垂体腺瘤侵袭性的研究

目的研究缺氧诱导因子-1α（HIF-1α）和血管内皮生长因子（VEGF）在人垂体腺瘤中的表达，探讨其表达水平与垂体腺瘤血管生成和侵袭性的关系。方法即用型免疫试剂盒检测23例垂体腺瘤中HIF-1α和VEGF的表达情况，比较两者表达的相关性；同时对表达水平与患者的临床资料进行统计学分析。结果HIF-1α和VEGF与垂体腺瘤的侵袭性密切相关（P〈0．05）。结论HIF-1α和VEGF可能通过促进新生血管形成刺激垂体腺瘤生长和侵袭，其作用机制有待进一步研究。     

Vascular endothelial growth factor plasma levels are significantly elevated in patients with cerebral arteriovenous malformations

BACKGROUND: Since growth and de novo generation of cerebrovascular malformations were demonstrated, a strictly congenital model cannot be further supported as unique factor in the pathogenesis of cerebral arteriovenous malformations (AVMs). Vascular endothelial growth factor (VEGF) has previously been demonstrated to be highly expressed in AVMs by immunohistochemical methods. However, systemic VEGF levels have not been analysed previously. This study aimed to investigate VEGF plasma concentrations as a possible plasma marker for neovascularization in patients with cerebral AVMs compared to healthy controls. METHODS: The study included 17 patients with cerebral AVMs and 40 healthy controls. VEGF plasma concentrations were measured by a specific enzyme immuno-assay. RESULTS: VEGF plasma concentrations were significantly higher in patients with cerebral AVMs (mean 140.9 pg/ml, SD 148.5 pg/ml and median 63.0 pg/ml) compared to a healthy control group (mean 44.7 pg/ml, SD 36.4 pg/ml and median 35.0 pg/ml), p = 0.0003. CONCLUSIONS: Our findings suggest that VEGF plasma concentrations might play a role in the pathogenesis of cerebral AVMs. Further studies are necessary and would contribute to an improved understanding of the pathogenesis of cerebral AVMs.     

核因子κB在低氧诱导因子1α基因修饰神经干细胞上调血管内皮生长因子表达中的桥接作用**

背景：很多研究发现,在脑缺氧损伤区域核因子κB与血管内皮生长因子表达呈正相关。因此,作者大胆假设,核因子κB是否处于低氧诱导因子1α上调血管内皮生长因子作用通路上并起桥接作用。 目的：以低氧诱导因子1α修饰的神经干细胞为载体,观察核因子κB 在低氧诱导因子1α上调血管内皮生长因子表达通路中的作用。 设计、时间及地点：细胞学体外观察,于2008-03/12在佳木斯大学神经科学研究所完成。 材料：新生24 h内的Wistar大鼠,雌雄不拘。 方法：扩增腺病毒载体低氧诱导因子1α-绿色荧光蛋白后转染神经干细胞,荧光检测神经干细胞中低氧诱导因子1α-绿色荧光蛋白及空载体Ad-绿色荧光蛋白表达,分别提取基因转染后神经干细胞、空载体转染神经干细胞、正常神经干细胞蛋白。然后低氧诱导因子1α基因修饰的神经干细胞中按50,150,300 μmol/L浓度梯度加入核因子κB特异性抑制剂二硫氨基甲酸酞吡咯烷,Western Blot法检测其中血管内皮生长因子的表达变化。 主要观察指标：各组神经干细胞中低氧诱导因子1α、血管内皮生长因子和核因子κB 的表达;给予梯浓度核因子κB 特异性抑制剂后神经干细胞中血管内皮生长因子的表达。 结果：腺病毒低氧诱导因子1α-绿色荧光蛋白转染神经干细胞后基因表达强弱与MOI及转染时间有关;转染低氧诱导因子1α-绿色荧光蛋白后的神经干细胞中低氧诱导因子1α、血管内皮生长因子和核因子κB的表达呈正相关;给予梯浓度核因子κB 特异性抑制剂后腺病毒低氧诱导因子1α-绿色荧光蛋白修饰的神经干细胞中血管内皮生长因子的表达呈抑制剂浓度依赖性下调,各浓度组之间血管内皮生长因子表达差异有显著性意义(P < 0.05~0.01)。 结论：核因子κB位于低氧诱导因子1α上调血管内皮生长因子表达的信号通路上并起桥接作用。     

Inflammatory molecule expression in cerebral arteriovenous malformations.

Inflammatory proteins may play a role in the pathophysiology of cerebral arteriovenous malformations and their response to radiosurgery. The aim of this study was to compare the expression of inflammatory molecules in arteriovenous malformations (AVMs) with that in normal cerebral vessels. Fresh-frozen surgical specimens from 15 AVMs and three control specimens were studied. The expression of P- and E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), platelet-endothelial cell adhesion molecule (PECAM-1) and von Willebrand factor were examined using immunohistochemistry. AVMs had significant upregulation of E-selectin. VCAM-1 and ICAM-1 upregulation was also observed in AVMs. Pre-operative embolization was associated with increased expression of E-selectin and VCAM-1. This study has provided further evidence that the endothelium of AVMs has different molecular properties than the endothelium of normal cerebral vasculature. Inflammatory molecules may be biologically relevant in the response of vascular malformations to radiosurgery and embolization.     

Hypoxia inducible factor-1 alpha stabilization for regenerative therapy in traumatic brain injury

Mild traumatic brain injury(TBI), also called concussion, initiates sequelae leading to motor deficits, cognitive impairments and subtly compromised neurobehaviors. While the acute phase of TBI is associated with neuroinflammation and nitroxidative burst, the chronic phase shows a lack of stimulation of the neurorepair process and regeneration. The deficiency of nitric oxide(NO), the consequent disturbed NO metabolome, and imbalanced mechanisms of S-nitrosylation are implicated in blocking the mechanisms of neurorepair processes and functional recovery in the both phases. Hypoxia inducible factor-1 alpha(HIF-1α), a master regulator of hypoxia/ischemia, stimulates the process of neurorepair and thus aids in functional recovery after brain trauma. The activity of HIF-1α is regulated by NO via the mechanism of S-nitrosylation of HIF-1α. S-nitrosylation is dynamically regulated by NO metabolites such as S-nitrosoglutathione(GSNO) and peroxynitrite. GSNO stabilizes, and peroxynitrite destabilizes HIF-1α. Exogenously administered GSNO was found not only to stabilize HIF-1α and to induce HIF-1α-dependent genes but also to stimulate the regeneration process and to aid in functional recovery in TBI animals.     

Expression levels of vascular endothelial growth factor and its receptors in Parkinson's disease

Recently, we confirmed the presence of enhanced neural reconstruction in Parkinson's disease and in an animal model of Parkinson's disease based on increased polysialic acid-like immunoreactivity. Changes in neurogenesis often appear parallel to changes in angiogenesis. Moreover, both these processes share similar modulating factors, like vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1). Using immunohistochemistry, we identified in this study upregulation of VEGF in the substantia nigra but not in the striatum of patients with Parkinson's disease by enzyme-linked immunosorbent assay. Such overexpression may participate in vascular remodeling and neurogenesis in the substantia nigra of Parkinson's disease.     

Hypoxia inducible factor-1alpha mediates protection of DL-3-n-butylphthalide in brain microvascular endothelial cells against oxygen glucose deprivation-induced injury

Studies have demonstrated that DL-3-n-butylphthalide can significantly alleviate oxygen glucose deprivation-induced injury of human umbilical vein endothelial cells at least partly associated with its enhancement on oxygen glucose deprivation-induced hypoxia inducible factor-1α expression.In this study,we hypothesized that DL-3-n-butylphthalide can protect against oxygen glucose deprivation-induced injury of newborn rat brain microvascular endothelial cells by means of upregulating hypoxia inducible factor-1α expression.MTT assay and Hoechst staining results showed that DL-3-n-butylphthalide protected brain microvascular endothelial cells against oxygen glucose deprivation-induced injury in a dose-dependent manner.Western blot and immunofluorescent staining results further confirmed that the protective effect was related to upregulation of hypoxia inducible factor-1α.Real-time RT-PCR reaction results showed that DL-3-n-butylphthalide reduced apoptosis by inhibiting downregulation of pro-apoptotic gene caspase-3 mRNA expression and upregulation of apoptosis-executive protease bcl-2 mRNA expression;however,DL-3-n-butylphthalide had no protective effects on brain microvascular endothelial cells after knockdown of hypoxia inducible factor-1α by small interfering RNA.These findings suggest that DL-3-n-butylphthalide can protect brain microvascular endothelial cells against oxygen glucose deprivation-induced injury by upregulating bcl-2 expression and downregulating caspase-3 expression though hypoxia inducible factor-1α pathway.     

HIF-1及NGB,VEGF在脑挫裂伤后促脑血管生成的作用机制

目的探讨HIF-1调控Ngb、VEGF在脑挫裂伤后促血管生成的作用。方法采用蛋白质组学技术比较脑挫裂伤患者和非颅脑创伤患者额叶组织的差异蛋白质,并应用生物信息学技术分析鉴定差异蛋白。然后运用免疫组化和酶联免疫方法分别检测差异蛋白HIF-1、Ngb及VEGF在脑组织以及血清中的表达。结果建立了脑挫裂伤患者和非颅脑创伤患者组脑组织的2-DE图谱,鉴定了7种差异蛋白,其中有HIF-1、Ngb及VEGF。HIF-1、Ngb及VEGF在脑挫裂伤患者脑组织中的表达水平均高于非颅脑创伤患者。HIF-1、Ngb及VEGF在脑挫裂伤患者血清中的表达水平均显著高于非颅脑创伤患者。结论脑挫裂伤后,HIF-1通过调控Ngb和VEGF两个靶基因的释放,在新的脑血管生成过程中发挥了重要作用。HIF-1,Ngb和VEGF在血清中的含量变化,可为临床评估和监测缺氧缺血脑病的严重程度提供参考;对脑挫裂伤的诊断、治疗以及疾病的转归具有重要意义。     

Thrombotic molecule expression in cerebral vascular malformations.

Thrombosis is an important end-point in the obliteration of vascular malformations after radiosurgery. The aim of this study was to investigate the expression of thrombotic molecules in arteriovenous malformations (AVMs) and cavernous malformations (CMs), and in AVMs after radiosurgery. Fresh-frozen surgical specimens from 18 AVMs (including three that had previously been treated with radiosurgery), seven CMs, and three control specimens were studied. The expression of tissue factor, thrombomodulin and von Willebrand factor (vWF) were examined using immunofluorescence. Thrombomodulin and vWF were expressed in the endothelium of all specimens, while tissue factor was predominately found in the perivascular region and vascular adventitia. Previous treatment of AVMs with either radiation or embolisation did not significantly alter the intensity of expression. In some irradiated lesions, vessels were found with absent endothelial vWF staining and exposed tissue factor. This study has demonstrated that loss of the endothelium and exposure of underlying tissue factor occurs in irradiated AVMs. There were no significant differences in the expression of these thrombotic molecules in vascular malformations when compared to control vessels. While no long-term alterations in antigen expression were observed after radiosurgery, further work may elucidate the nature of the immediate response to irradiation.     

Expression of platelet-derived growth factor ligand and receptor in cerebral arteriovenous and cavernous malformations

The aim of this study was to investigate the expression of platelet-derived growth factor (PDGF) ligands A and B and receptors α and β in cerebral arteriovenous and cavernous malformations. Fifteen arteriovenous malformation (AVM) and 15 cerebral cavernous malformation (CCM) tissue samples were immunostained for PDGF ligands A and B, PDGF receptors (PDGFR) α and β, and vascular endothelial growth factor. Tissues were compared in terms of expression levels within various vascular layers, and the results were confirmed using western blotting. AVM had higher levels of PDGF-A expression than CCM (p = 0.004, 0.009, 0.001, and 0.027, for endothelium, media, adventitia, and perilesional tissue, respectively) and western blotting showed that there was higher expression of PDGFR-α in AVM tissues. In contrast, CCM endothelium, media, and adventitia had higher PDGF-B expression compared with AVM (p = 0.007, 0.001, and 0.039, respectively). PDGFR-β expression was also significantly higher in the endothelium of CCM tissue (p = 0.007). Overexpression of PDGF ligands and receptors in AVM and CCM may mean that therapeutic strategies targeting the PDGF pathway could be useful in the treatment of these two malformations.     

Vascular endothelial growth factor expression in cerebral neoplasms

Angiogenesis plays an important role in growth of neoplasm. Among a variety of proangiogenic agents, vascular endothelial growth factor (VEGF) is regarded as a crucial mediator of tumour angiogenesis. It acts in a paracrine way through the receptors localised in endothelial cells. Many authors maintain that rich vasculature of the neoplasm is associated with its malignant nature. The aim of this study was to examine the relations between expression of VEGF and features of malignancy of brain tumours. Sixty-seven samples of brain tumours were examined: 17 meningiomas, 34 gliomas and 16 metastases to the central nervous system. Expression of VEGF was estimated by radioimmune assay. The authors confirmed the presence of this factor in all types of tumours but the highest concentration of VEGF was found in high-grade gliomas.     

血管内皮生长因子表达载体的构建及在内皮祖细胞中的表达

背景：血管内皮生长因子(vascular endothelial growth factor, VEGF)具有很强的促进血管生成作用，但构建其真核表达质粒是否可转染血管内皮祖细胞并完整表达还不清楚。 目的：构建VEGF表达载体,并观察其在内皮祖细胞中的表达情况。 设计、时间及地点：观察性试验，于2007-12/2008-03在上海交通大学医学院附属新华医院科研中心实验室完成。 材料：质粒PDC315-VEGF165自备，质粒pIRES2-EGFP由美国国立卫生研究院Stanko Stojilkovic教授惠赠。 方法：运用DNA重组技术构建VEGF表达载体pIRES2-EGFP-VEGF，应用脂质体包裹的方法将载体转染猪外周血血管内皮祖细胞。 主要观察指标：采用荧光显微镜观察VEGF在内皮祖细胞内的表达，反转录-聚合酶链反应法检测VEGFmRNA表达水平， ELISA检测VEGF165蛋白表达情况。 结果：成功构建VEGF真核表达载体pIRES2-EGFP-VEGF。转染重组质粒pIRES2-EGFP-VEGF后,在内皮祖细胞内有VEGF的表达，VEGFmRNA和VEGF165 蛋白表达水平均明显增加。 结论：VEGF 表达载体转染猪外周血血管内皮祖细胞后能有效增加VEGF基因的表达，能够获得较高水平的VEGF蛋白。     

Puerarin decreases hypoxia inducible factor-1 alpha in the hippocampus of vascular dementia rats

In this study, a rat vascular dementia model was established by permanent bilateral common carotid arterial occlusion. Rats were intraperitoneally injected with puerarin 3 days before modeling, for 45 successive days. Results demonstrated that in treated animals hippocampal structures were clear, nerve cells arranged neatly, and cytoplasm was rich in Nissl bodies. The number of cells positive for hypoxia inducible factor-1 alpha, erythropoietin and endothelial nitric oxide synthase was reduced; and the learning and memory abilities of rats were significantly improved. Our experimental findings indicate that puerarin can significantly improve learning and memory in a vascular dementia model, and that the underlying mechanism may be associated with the regulation of the expression of hypoxia inducible factor-1 alpha.     

Vascular endothelial growth factor and vascular endothelial growth factor receptor-2 expression in mdx mouse brain

Recent data have demonstrated that vascular endothelial growth factor (VEGF) is expressed by subsets of neurons, coincident with angiogenesis within its developing cerebral cortex. In this study, with the aim of elucidating the mechanisms of vascular involvement during brain impairment in Duchenne muscular distrophy (DMD), we have correlated the vascular density with VEGF and VEGF receptor-2 (VEGFR-2) expression in the brain cortex of normal and mdx mouse, an animal model with a genetic defect in a region homologous with the human DMD gene. Results showed that in mdx mouse, tissue area occupied by microvessels positive to factor VIII related antigen and VEGFR-2 increased in parallel to the tissue area occupied by neurons positive to VEGF. Our data suggest that increased vascularity in the brain of mdx mouse may be due, at least in part, to proliferation of endothelial cells in response to VEGF secreted by neuronal cells.     

可溶性血管内皮细胞生长因子受体1真核表达载体pEGFP-N1/sflt-1构建及在人脐静脉内皮细胞中的表达

背景：研究表明,可溶性血管内皮生长因子受体Flt-1(sflt-1)基因在多种组织中表达,但其在内皮细胞中的表达尚待证实。 目的：获取人sflt-1基因,构建sflt-1基因真核表达载体pEGFP-N1/sflt-1重组质粒,检测其在人脐静脉内皮细胞中的表达。 设计、时间及地点：细胞学体外观察,于2008-05/12在南昌大学第二附属医院分子中心完成。 材料：pEGFP-N1真核表达载体为南昌大学第二附属医院分子中心保存,人脐静脉内皮细胞株购至南京基凯生物技术公司。 方法：从含有目的基因的质粒克隆模板中,利用酶切的方法获得目的基因,将目的载体进行酶切,纯化酶切产物后进行定向连接,其产物转化细菌感受态细胞,PCR鉴定为阳性的克隆,证明目的基因已经定向连入目的载体。再对PCR鉴定阳性的克隆进行测序和分析比对,比对正确的即为构建成功的融合蛋白表达质粒载体。用脂质体法将pEGFP-N1/sflt-1重组质粒转染人脐静脉内皮细胞。 主要观察指标：①重组质粒的酶切鉴定结果。②重组质粒的测序鉴定结果。③荧光显微镜及蛋白免疫印迹检测sflt-1基因在人脐静脉内皮细胞中的表达。 结果：①对PCR鉴定阳性的克隆进行测序和分析比对,与sflt-1基因片段全长2 063 bp理论值相符,证实pEGFP-N1/sflt-1重组质粒构建成功。②重组质粒送至上海捷瑞生物技术公司进行通测,测序结果证实插入片段序列与理论值完全一致。③荧光显微镜下可见重组质粒转染人脐静脉内皮细胞发绿色荧光,蛋白免疫印迹检测表明sflt-1基因能在人脐静脉内皮细胞中表达。 结论：实验成功构建了携带人血管内皮生长因子受体Flt-1的重组pEGFP-N1/sflt-1真核表达质粒,并证实其在人脐静脉内皮细胞中的表达。