血管内皮细胞特异性生长因子与胶质瘤血管生成

实体性肿瘤是血管依赖性的,肿瘤细胞需要新生血管带来氧和养料及带走代谢产物维持其较高的代谢率,如果肿瘤血管生成不能满足肿瘤生长的需要,肿瘤细胞会发生坏死或凋亡.肿瘤血管生成包括内皮细胞的增殖、迁移、细胞外基质溶解及募集周围细胞形成成熟血管,涉及多种生长因子及细胞因子参与.     

胶质瘤的抗血管生成治疗现状

胶质瘤是最常见的恶性肿瘤之一。血管的生成及其生长速度对肿瘤生长、侵袭和转移起着关键性的作用,近年的研究证实,胶质瘤组织内的血管生成与血管内皮生长因子(VEGF)与其恶性程度及预后等具有密切联系;因此,抗血管生成治疗目前已成为传统化疗方法之外的胶质瘤治疗新策略。现就脑胶质瘤的抗血管生成治疗进展进行综述。     

胶质瘤抗肿瘤血管生成治疗的新对策和新途径——抑制胶质瘤血管内皮生长因子的表达

实体瘤是典型的血管依赖性病变 ,血管内皮生长因子 (VEGF)是肿瘤血管生成的关键因素。胶质瘤是一种富含血管的恶性肿瘤 ,胶质瘤的血管内皮增生随恶性程度增加而明显增加 ,故VEGF是胶质瘤抗肿瘤血管生成的良好靶标 ,通过抑制及阻断血管内皮生长因子 (VEGF)的表达和作用来抑制胶质瘤血管生成是与常规治疗方法不同的一种治疗新途径及新对策。     

叶酸/聚酰胺一胺介导miR -7抑制胶质瘤U251恶性表型的研究

目的 研究叶酸/聚酰胺-胺( FA/PAM AM)作为miR -7载体治疗胶质瘤U251的效果。方法将FA/PAMAM/miR -7络合物转染胶质瘤细胞株U251,绘制细胞生长曲线,检测细胞周期和细胞凋亡情况,Transwell体系检测瘤细胞迁移能力,软琼脂克隆实验检测细胞致瘤性;qRT - PCR方法检测miR -7水平,Western blot方法检测EGFR、PI3K和AKT2的蛋白表达。结果 获得稳定转染的U251细胞增殖速度减慢,细胞周期延长,凋亡细胞比例增多,细胞迁移能力、软琼脂克隆形成能力均有明显降低(P＜0.05);miR -7水平明显提高,EGFR、PI3K和AKT2蛋白水平均有一定程度降低(P＜0.05)。结论 FA/PAMAM能够高效投递miR -7基因至胶质瘤细胞,发挥肿瘤生长抑制作用。     

恶性胶质瘤抗血管生成治疗现状

血管生成,即从已存在的微血管系统中生成新的血管,在生理状态及肿瘤生长过程中均可发生。在生理状态下,血管生成过程在胚胎发育、胎儿出生后及女性生殖周期中是不可或缺的。而且,血管     

靶向表皮生长因子受体psiRNA抑制人脑胶质瘤细胞系U251生长的体外研究

目的探讨靶向表皮生长因子受体(EGFR)RNA干扰(RNAi)技术抑制人脑胶质瘤细胞系U251EGFR表达后,在体外对细胞增殖活性和侵袭能力的抑制作用。方法应用流式细胞术、Annexin V法、MTT法、Matrigel细胞外基质生长实验和Transwell细胞侵袭实验评价由脂质体Lipofectamine 2000介导转染的人脑胶质瘤细胞系U251转染前后细胞增殖活性和侵袭能力的变化;Western blotting检测EGFR和PI3K/Akt信号转导通路中相关蛋白质的表达水平。结果与对照组和psiRNA-scr组比较,psiRNA-EGFR组EGFR以及PI3K/Akt信号转导通路中相关蛋白质表达水平降低(均P=0.000)。转染后,psiRNA-EGFR组细胞阻滞于G0和G1期,S期细胞数目减少(均P=0.000);其细胞凋亡率为(9.60±0.26)%,高于对照组和psiRNA-scr组(均P=0.000);自培养第2天其细胞增殖速率即呈下降趋势,且低于对照组和psiRNA-scr组(均P=0.001)。结论靶向表皮生长因子受体RNAi技术可以序列特异性地抑制人脑胶质瘤细胞系U251细胞表皮生长因子受体表达,在体外对U251细胞生长具有明显抑制作用。靶向表皮生长因子受体psiRNA基因治疗可能成为脑胶质瘤治疗的新策略。     

胶质瘤中促血管生成素2和血管内皮生长因子基因表达的意义

目的 探讨胶质瘤促血管生成素（Ang2）和血管内皮细胞生长因子（VEGF）基因表达的意义。方法 采用逆转录-酶促链反应（RT-PCR）检测52例人脑胶质瘤中Ang2、VEGF基因的表达，同时采用SABC免疫组化方法检测Ang2蛋白的表达。结果 50例脑胶质瘤表达Ang2 mRNA片段．52例脑胶质瘤表达VEGF mRNA片段。Ang2 mRNA与VEGF mRNA表达呈显著性正相关（r=0，816，P〈0．01）；高度恶性胶质瘤Ang2mRNA、VEGF mRNA表达均显著性高于低度恶性者（P〈0．01）。免疫组化染色显示Ang2蛋白主要分布在高度恶性胶质瘤组织的瘤细胞和内皮细胞．而在低度恶性胶质瘤中呈低水平表达。结论 Ang2与VEGF可能在胶质瘤血管生成中起协同性促进作用。     

恶性胶质瘤靶向治疗从实验室走向临床的范例：抗肿瘤血管生成治疗

恶性胶质瘤的治疗是神经外科领域毋庸置疑的挑战。尽管临床医生不断探索和革新手术切除以及化学治疗和放射治疗方案,但传统的综合治疗措施在延长恶性胶质瘤患者生存期方面似乎已达“极限”。新的治疗思路必须向基础研究寻求答案。恶性胶质瘤分子生物学与分子遗传学研究历经十余年的积累,现有结论已经告诉我们,恶性胶质瘤发生和发展涉及多基因异常,这些基因多属于信号转导通路中的重要成分,     

粘附分子与胶质瘤血管生成

在肿瘤的血管生成过程中,粘附分子起着极其重要的作用。其作用大体可以概括为:诱导内皮细胞迁移至血管生成部位;引起内皮细胞的增殖;介导内皮细胞以及内皮细胞和周细胞(Pericyte)之间的粘附连接;形成毛细血管;细胞之间信号的传导等等。胶质瘤具有丰富的血管,在胶质瘤的血管生成过程中粘附分子同样起重要作用。通过作用于粘附分子可以抑制肿瘤的血管生成,达到治疗胶质瘤的目的。     

CXCR4在胶质瘤诱导血管生成中的作用

目的 研究CXCR4在人脑胶质瘤中的表达及其与肿瘤恶性程度、微血管密度(MVD)之间的关系,进一步探讨CXCR4在胶质瘤血管生成中的作用及CXCR4作为抗肿瘤靶点的可能性.方法 收集河南省人民医院神经外科自2009年9月至2011年9月手术切除的人脑胶质瘤标本41例,其中低度恶性胶质瘤(Ⅰ～Ⅱ级)18例,高度恶性胶质瘤(Ⅲ～IV级)23例.另外取行内减压术的脑外伤患者的新鲜脑组织10例作为对照.免疫组织化学染色检测标本CXCR4和CD34的表达,实时荧光定量PCR检测标本CXCR4mRNA的表达.结果 免疫组化染色结果显示对照组CXCR4表达阴性,脑胶质瘤组织CXCR4表达阳性28例(68.29％),脑胶质瘤组织MVD的表达高于对照组,且高度恶性胶质瘤组CXCR4表达阳性率、MVD的表达均高于低度恶性胶质瘤组,差异有统计学意义(P＜0.05);CXCR4表达阳性组MVD(42.51±6.99)明显高于CXCR4表达阴性组(25.98±4.99),差异有统计学意义(P＜0.05);高度恶性胶质瘤组CXCR4mRNA的表达量(5.19±1.43)高于低度恶性胶质瘤组(2.28±0.71),差异有统计学意义(P＜0.05).结论 CXCR4在胶质瘤血管生成中发挥重要的作用,有可能成为胶质瘤抗血管生成治疗的新靶点.     

靶向AKT1和COX-2的RNAi抑制U251胶质瘤细胞侵袭的体外实验

目的应用RNA干扰(RNA interference,RNAi)技术敲低恶性胶质瘤细胞系U251中AKT1和COX-2表达后,在体外对细胞侵袭转移的抑制作用,初步探讨其可能的作用机制。方法构建重组腺病毒载体rAd5-A-C同时搭载靶向AKT1和COX-2的shRNA干扰序列,将其转染至恶性胶质瘤细胞系U251细胞。Realtime PCR检测RNAi后目的基因mRNA的表达水平,Western Blot检测目的基因及MMP-2、MMP-9、TIMP-2的表达情况。酶联免疫吸附试验检测转染前后分泌到细胞外的MMP-2和MMP-9的浓度变化。划痕实验和Transwell实验评价肿瘤细胞转染前后的细胞侵袭能力的变化。结果rAd5-A-C转染组AKT1和COX-2的mRNA和蛋白表达均明显抑制;MMP-2、MMP-9表达下调,TIMP-2表达则上调。ELISA检测胞外MMP-2、MMP-9浓度明显减低;划痕实验显示细胞转移运动能力明显减弱,Transwell体外侵袭实验结果显示穿过细胞数明显减低(P0.001)。结论重组腺病毒介导的RNAi技术可以序列特异性地抑制U251细胞AKT1和COX-2的表达,在体外对U251细胞侵袭转移产生明显抑制作用,可能成为恶性胶质瘤治疗的新策略。     

血管内皮生长因子受体2小干扰RNA抑制内皮祖细胞血管新生

目的 探讨血管内皮生长因子受体2(vascular endothelial growth factor receptor 2,VEGFR-2)在内皮祖细胞(endothelial progenitor cells,EPCs)血管新生中的作用.方法 通过VEGFR-2小干扰RNA(small interfering RNA,siRNA)转染EPCs,检测其转染效率与抑制效率,观察EPCs体外血管生成以及迁移能力,并分析其基质金属蛋白酶-9(matrix metalloproteinases-9,MMP-9)、丝氨酸/苏氨酸激酶(serine threonine,Akt)、胞外信号调节激酶(extracellular signal-regulated kinase,ERK)的蛋门表达.结果 VEGFR-2 siRNA能有效并特异地抑制EPCs VEGFR-2的表达;VEGFR-2 siRNA能抑制EPCs的体外血管生成与迁移能力;VEGFR-2 siRNA能抑制EPCs MMP-9的表达与活性,以及Akt与ERK的磷酸化水平.结论 VEGFR-2 siRNA可能通过下调MMP-9、Akt与ERK的表达来抑制EPCs血管新生.     

Knockdown of annexin 2 decreases migration of human glioma cells in vitro

Diffuse invasion of brain tissue is a major reason for the poor prognosis of patients with glioblastoma. Annexin 2, a member of the large annexin family of Ca2+ and membrane-binding proteins, is expressed at high protein levels in human gliomas and has been proposed as a marker of glioma malignancy, while its functional role in these tumours is unknown so far. The ability of annexin 2 to interact with the actin cytoskeleton, as well as its potential to bind invasion-associated proteases, suggests that it could participate in invasion-associated processes in human gliomas. Therefore, we analysed here functional consequences of RNA interference-mediated silencing of annexin 2 in U87MG and U373MG human glioma cell lines. While no impact of annexin 2 downregulation on proliferation and adhesion was observed, our analyses revealed that migration of U87MG and U373MG cells was significantly inhibited following annexin 2 depletion. This effect was not related to a compensatory increase of the related annexins 1 or 6. Our findings identify annexin 2 as a potential candidate involved in glioma invasion and support the potential of RNA interference as powerful tool in the decryption of glioma invasion mechanisms.     

姜黄素诱导自噬抑制脑胶质瘤U87细胞增殖的研究

目的研究姜黄素对脑胶质瘤U87细胞增殖的抑制作用及机制。方法采用10、20、40、60、80μmol/L姜黄素分别处理U87细胞72h后,MTT法检测姜黄素对U87细胞增殖的影响。流式细胞术检测40μmol/L姜黄素处理前后U87细胞周期和凋亡变化。提取40μmol/L姜黄素处理前后不同时间点U87细胞总蛋白,Westernblot检测自噬相关蛋白LC3的表达。结果姜黄素呈剂量依赖性抑制U87细胞增殖(P0.05),半数抑制浓度(IC50)为42.44μmol/L。姜黄素诱导G2-M期细胞周期阻滞,实验组G2-M期细胞比例为(47.71±2.67)%,对照组为(17.96±0.88)%(P0.01);实验组细胞凋亡率为(3.93±0.82)%,对照组为(1.80±0.65)%,两组差异无统计学意义(P0.05)。姜黄素给药12、24、48h,LC3-Ⅱ/LC3-Ⅰ比值分别为(0.89±0.004)、(0.93±0.01)、(1.09±0.02),高于对照组(0.76±0.04)(P0.05);3-MA能减弱姜黄素对U87细胞增殖的抑制作用。结论姜黄素通过诱导自噬抑制脑胶质瘤U87细胞的增殖。     

血管内皮生长因子反义RNA对C6鼠胶质瘤细胞体外作用的研究

目的探讨VEGF反义RNA对C6鼠胶质瘤细胞体外作用。方法用Lipofectamine介导的方法将VEGF反义eDNA转染入C6鼠胶质瘤细胞系,观察其对细胞增殖和凋亡的影响。结果在体外VEGF反义RNA可有效抑制C6鼠胶质瘤细胞内源性VEGFmRNA和VEGF蛋白的产生,但对细胞增殖和凋亡无明显影响。结论采用VEGF反义RNA可能成为抗血管形成肿瘤治疗的新策略之一。     

蒿甲醚对C6胶质瘤细胞的抑制作用

目的 研究蒿甲醚对大鼠C6胶质瘤细胞的抑制作用.方法 C6胶质瘤细胞经培养后,按是否加入蒿甲醚分为实验组和对照组,均采用四甲基偶氮唑盐(MTT)法、流式细胞技术(FCM)及Hoechst33258荧光染色法检测细胞凋亡情况.结果 MTT法检测显示:24、48、72h3个时间组蒿甲醚对C6胶质瘤细胞的半数抑制浓度(IC50)分别为(195.08±3.27) μmol/L、(119.64±4.06) μmol/L、(87.84±0.93) μmol/L.FCM法检测显示:24、48、72 h3个时间组C6胶质瘤细胞凋亡率分别为:7.95％、22.01％、31.22％;且G0-G1期细胞比例增加,S期和G2-M期细胞比例降低.荧光染色显示:实验组细胞内出现凋亡小体;而对照组细胞呈弥散均匀荧光.结论 蒿甲醚能抑制C6胶质瘤细胞生长,且其抑制作用呈现时间依赖性和浓度依赖性;蒿甲醚能干扰C6胶质瘤细胞的细胞周期,可将其阻滞在G0-G1期并诱导其凋亡.     

Inhibitory effect of calcium channel blockers on proliferation of human glioma cells in vitro

The effects of 2 specific calcium channel blockers, verapamil and nimodipine, on the proliferation of human glioma tumour cells were investigated in vitro. Tumour tissues for primary cell cultures were obtained bioptically from 3 patients with the histopathological diagnosis of glioblastoma. The [3H]-thymidine incorporation into glioma tumour cells DNA was used as a sensitive index of the cell proliferation. It was found that verapamil (10(-4)-10(-5) M) and nimodipine (10(-4)-10(-6) M) significantly inhibited the [3H]-thymidine uptake in a dose-related manner. The inhibitory effect of both calcium channel antagonists was reversed by simultaneous addition of calcium chloride (5 x 10(-3) M). These results indicate that verapamil and nimodipine may exert an antiproliferative effect on glioma cells growth acting through a blockade of specific voltage-dependent calcium channels.     

CXCR4活化促进人恶性胶质瘤细胞分泌血管生成因子

目的观测CXCR4活化对人恶性胶质瘤细胞系U87中血管内皮生长因子（VEGF）、白细胞介素-8（IL-8）基因表达及蛋白分泌的影响。方法采用间接免疫荧光标记观测CXCR4在U87细胞中的表达和定位；用间质细胞衍生因子-1α（SDF-1α）激活CXCR4，通过酶联免疫吸附试验（ELISA）检测U87细胞培养上清中VEGF、IL-8蛋白的含量，用逆转录聚合酶链反应（RT-PCR）检测二者mRNA的表达。结果CXCR4表达于U87细胞的胞膜和胞质，经活化后可增加VEGF、IL-8mRNA的表达和蛋白分泌量。结论人恶性胶质瘤细胞中CXCR4活化后能够促进血管生成因子VEGF和IL-8的产生。     

Expression of the B7-related molecule ICOSL by human glioma cells in vitro and in vivo

Human glioblastoma is a highly lethal tumor known for its capability of interfering with effective antitumor immune responses. Costimulatory signals are of critical relevance in both the inductive and effector phases of immune responses. Inducible costimulator-ligand (ICOSL), a member of the B7 family of costimulatory molecules related to CD80/CD86, regulates CD4 as well as CD8 T-cell responses via interaction with its receptor, ICOS, on activated T cells. We report the expression of ICOSL by glioma cells in vitro and in vivo. In contrast to CD80 (B7.1) and CD86 (B7.2), ICOSL protein and mRNA was expressed in 7 of 12 glioma cell lines. ICOSL expression is upregulated by the inflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), whereas interferon-gamma (IFN-gamma) has no such effect. Further, immunohistochemical analysis of human brain tumors demonstrates the expression of ICOSL in three of four tissue samples. ICOSL expression is functional in that a neutralizing ICOSL antibody (HIL-131) reduces Th1 and Th2 cytokine levels in cocultures of peripheral blood lymphocytes or T-cell subsets (CD4 and CD8) with glioma cells. However, ICOSL gene transfer into glioma cells does not alter their immunogenicity under primary or secondary alloreactive coculture assays.     

Effects of Raf-1 siRNA on human cerebral microvascular endothelial cells: a potential therapeutic strategy for inhibition of tumor angiogenesis

The serine/threonine kinase Raf-1 is involved in the regulation of tumor cell survival, proliferation and metastasis formation, and has therefore emerged as a promising target for cancer therapy. In addition, Raf-1 activity mediates proliferation of endothelial cells thereby promoting angiogenesis and invasive growth of various tumors, including highly vascularized malignant glioblastoma. The aim of this study was to evaluate the effects of small inhibitory RNA (siRNA) directed against Raf-1 on viability, proliferation and motility in glioma cells and cerebral endothelial cells. Half-quantitative RT-PCR and Western blotting revealed efficient siRNA-mediated Raf-1 down regulation in glioma cells (U373, U251) and in human cerebral microvascular endothelial cells (HCMEC). Surprisingly, Raf-1 gene silencing failed to affect cell survival, proliferation or migration activity in the glioblastoma cell lines. In HCMEC, however, pronounced decrease of cell survival and significant inhibition of tube formation was achieved by Raf-1 siRNA compared to non-functional siRNA or vehicle controls. In conclusion, Raf-1 silencing appears as a potential therapeutic strategy to inhibit brain tumor angiogenesis and thereby outgrowth of highly vascularized glioblastoma multiforme, whereas direct cytotoxic effects of Raf-1 knockdown in tumor cells may vary.