恶性胶质瘤细胞甲酰化肽受体表达及其活化后促瘤细胞增殖和血管生成因子产生的作用

背景与目的:趋化因子受体在多种病理过程中发挥重要作用,通过促进肿瘤细胞增殖、迁移或血管生成过程,进而在肿瘤发生、演进和转移中发挥极为重要的作用.本研究拟探讨甲酰化肽受体(formylpeptide receptor,FPR)在人恶性胶质瘤细胞中的表达和激活后的功能活性.方法:以人恶性胶质瘤细胞U87和FPR-siRNA-U87细胞为研究对象,采用间接免疫荧光标记、激光共聚焦扫描显微术观测人恶性胶质瘤细胞FPR的表达和定位;用FPR配体甲酰-甲硫酰-亮氨酰-苯丙氨酰胺(formy-Met-Leu-Phe,fMLF)激活FPR后,采用四甲基偶氮唑蓝(MTT)法检测细胞增殖,双抗夹心酶联免疫(ELISA)吸附法检测细胞分泌血管内皮生长因子(vascular endothelial growth factor,VEGF)和白细胞介素8(interleukin-8,IL-8)水平,RT-PCR法检测细胞IL-8 mRNA的水平.结果:恶性胶质瘤细胞U87有FPR表达,定位于细胞膜,FPR-siRNA-U87细胞不表达FPR:FPR激动剂fMLF对人恶性胶质瘤细胞具有明显的促增殖作用,U87细胞fMLF刺激组A值较对照组显著升高(P＜0.05),FPR-siRNA-U87细胞fMLF刺激组A值与对照组相比无显著性差异(P＞0.05);fMLF作为FPR的激动剂,在12 h、24 h、36 h和48 h等各时相点U87细胞的VEGF和IL-8蛋白水平均显著增加,以处理36 h为观察时间点,U87细胞VEGF蛋白水平[(3.13±0.23)ng/ml]较对照组显著增加(P＜0.05),IL-8蛋白分泌量为(7.54±0.53)ng/ml,较对照组显著增加(P＜0.05);FPR-siRNA-U87细胞VEGF和IL-8蛋白分泌量分别为(1.26±0.05)ng/ml和(1.54±0.05)ng/ml,较对照组无明显改变(P＞0.05);fMLF能显著上调U87细胞IL-8 mRNA的表达,较对照组显著升高(P＜0.05),FPR-siRNA-U87细胞IL-8 mRNA水平与对照组相比无显著性差异(P＞0.05).结论:人恶性胶质瘤细胞膜存在功能性FPR受体,其活化后具有促瘤细胞增殖和分泌血管生成因子的作用.     

MiR-29a下调共刺激分子B7-H3的表达及其对脑胶质瘤细胞侵袭的影响

目的： 探讨miR-29a调控共刺激分子B7-H3在脑胶质瘤中的表达及其对脑胶质瘤细胞侵袭能力的影响。 方法： 通过Real-time PCR检测miR-29a和B7-H3在正常脑组织、脑胶质瘤组织及人胶质瘤细胞株U87中的表达,并利用脂质体将miR-29a的模拟物（mimics）和抑制剂（inhibitors）转入U87细胞,流式术验证miR-29a对B7-H3表达的调节效果;采用CCK-8和Transwell实验观察miR-29a对U87细胞的增殖和侵袭能力的影响,并通过流式术分析miR-29a干预前后U87细胞上与细胞侵袭相关的化学趋化因子的表达变化,以miRtarbase等软件预测miR-29a与 CXCR4 的结合能力。 结果： 胶质瘤组织及细胞株中miR-29a低表达而 B7-H3 mRNA高表达,且均与瘤组织病理分级相关。转染miR-29a mimics可以有效下调U87细胞中 B7-H3 mRNA的表达。转染miR-29a mimics可以显著抑制U87细胞的侵袭能力（P<0.05）,但对细胞增殖并无显著影响。miR-29a过表达可同时下调U87细胞中CXCR4的表达,但软件分析 CXCR4 基因上并不存在miR-29a的结合位点。 结论： miR-29a可有效下调B7-H3分子的表达,进而抑制脑胶质瘤细胞的侵袭能力,其作用机制可能与CXCR4途径相关。     

趋化因子受体CXCR4活化促进人胶质瘤干细胞迁移及其机制探讨

目的:研究趋化因子受体CXCR4活化对胶质瘤干细胞(glioma stem cells, GSCs)迁移能力的影响,并探索其下游相关信号通路.方法:采用逐渐添加神经干细胞培养液的方法从人胶质细胞瘤U87细胞株中获得GSCs,利用Transwell小室法评估CXCR4活化对GSCs迁移能力的影响,并进一步应用Western印迹法观察CXCR4下游信号通路的激活情况.结果:CXCR4的配体CXCL12以浓度依赖的方式促进GSCs迁移,同时伴有Akt磷酸化增加;CXCR4特异性拮抗剂AMD3100能够抑制此效应.PI3K/Akt通路抑制剂LY294002可通过抑制Akt磷酸化而抑制CXCL12诱导的GSCs迁移.结论:CXCR4活化后激活其下游的PI3K/Akt信号通路,进而促进GSCs迁移,这可能是GSCs高侵袭特性的机制之一.     

CCL18通过与Nir1结合促进胶质瘤细胞侵袭的研究

背景与目的：Nir1是CCL18的跨膜受体,CCL18能与之特异性结合而促进乳腺癌细胞的侵袭与转移,但其在胶质瘤细胞中的作用尚不清楚。该文旨探讨Nir1在胶质瘤侵袭中的作用及分子机制。方法：应用蛋白［质］印迹法(Western blot)检测Nir1在不同胶质瘤细胞系中的表达;利用siRNA技术抑制U251细胞中Nir1的表达;采用Western blot检测转染后Nir1蛋白的表达和转染前后U251细胞中Akt磷酸化的情况;使用体外侵袭实验检测转染后细胞的运动和侵袭能力;采用F-actin聚合实验检测F-actin的聚合能力。结果：Nir1在各胶质瘤细胞中均呈高表达;小RNA干扰技术沉默Nir1基因后,U251细胞中Nir1的蛋白表达量明显下降,侵袭并穿透Matrigel胶的细胞数目明显比对照组少(P=0.00);在CCL18刺激后细胞内的F-actin聚合量比对照组减少;Akt磷酸化试验结果显示,对照组细胞在CCL18的刺激下Akt更易发生磷酸化,实验组细胞无论是否存在CCL18,Akt磷酸化都受到抑制。结论：在胶质瘤细胞中存在Nir1蛋白高表达,通过与细胞膜上CCL18特异性结合来调节胶质瘤细胞的F-actin聚合和Akt的磷酸化进而调控胶质瘤细胞的运动和侵袭能力。     

血管内皮细胞通过Notch信号通路对胶质瘤细胞侵袭性生长的影响

目的 探讨血管内皮细胞对胶质瘤细胞侵袭力的影响及其与Notch信号通路之间的关系。 方法 在体外采用Transwell共培养系统将人脐静脉内皮细胞（human umbilical vein endothelial cells, HUVEC）和U87胶质瘤细胞进行间接共培养;抑制剂DAPT（N-[N-(3,5-di-fluorophenacetyl)-L-alanyl]- S-phenylglycinet-butylester）抑制Notch信号通路; Transwell小室快速检测各组U87细胞的侵袭力;Real-time PCR检测各组U87细胞中Notch1、Notch2、Hes1、Delta样配体4（Dll4）、Cathepsins B、MMP-9、MMP-2 mRNA的表达情况;Western blot检测各组U87细胞中MMP-9、MMP-2蛋白、Cathepsins B的表达情况。结果 共培养组较单纯组U87细胞生长旺盛,DAPT处理后生长受抑制;共培养组穿过Matrigel膜的U87细胞数目明显多于单纯组,DAPT处理组较共培养组穿膜细胞数目减少。共培养组U87细胞的Notch1、Notch2、Hes1、Dll4、Cathepsins B、MMP-9 mRNA的表达及各组U87胶质瘤细胞中MMP-9、Cathepsins B蛋白的表达水平明显升高,DAPT处理组较共培养组相关的mRNA和蛋白水平降低。结论 血管内皮细胞可以通过Notch信号通路增强U87胶质瘤细胞的侵袭力,DAPT抑制Notch信号通路可以降低血管内皮细胞对U87胶质瘤细胞侵袭力的增强效果,Notch信号通路在血管内皮细胞对U87胶质瘤细胞的侵袭力的增强过程中起重要的调控作用。     

5-Aza-CdR对人脑胶质瘤细胞miR-129-2表达水平及其生物学行为的影响

目的:探讨DNA甲基转移酶抑制剂5-氮杂-2'-脱氧胞苷（5-Aza-CdR）对人脑胶质瘤细胞U87和U373中miR-129-2表达水平及细胞增殖、侵袭迁移能力的影响。方法:以0.5 mmol/L的5-Aza-CdR处理人脑胶质瘤细胞U87和U373 72 h,采用RT-PCR法检测miR-129-2的表达水平,CCK-8法观察胶质瘤细胞增殖能力,Transwell试验检测细胞的侵袭能力,划痕实验检测细胞的迁移能力。结果:经5-Aza-CdR处理后人脑胶质瘤细胞U87和U373的miR-129-2表达水平显著上调,细胞增殖能力、侵袭迁移能力受到抑制。结论:5-Aza-CdR能使miR-129-2启动子去甲基化,使其表达上调,并能抑制胶质瘤细胞增殖、侵袭迁移能力。     

敲减lncRNA RP1-261G23.7抑制胶质瘤细胞增殖和迁移及其机制探讨北大核心

目的:探讨长链非编码RNA(long non-coding RNA,lncRNA)RP1-261G23.7对人胶质瘤细胞增殖及迁移的影响及可能的调控机制。方法:采用GEPIA数据库分析RP1-261G23.7在胶质瘤组织中的相对表达。采用qRT-PCR检测RP1-261G23.7在4种胶质瘤细胞系(U87MG、SNB-19、U251、LN382)中的相对表达。采用Lipofectamine3000向胶质瘤细胞U87MG中单独转染si-RP1-261G23.7质粒(si-RP1-261G23.7组)和si-NC对照质粒(si-NC组)。采用CCK-8实验和细胞划痕实验检测转染后U87MG细胞的增殖及迁移能力。采用生物信息学、qRT-PCR和双荧光素酶报告基因实验研究RP1-261G23.7和miR-525-5p表达的关系。Western blotting检测NF-κB信号通路蛋白的表达。结果:与正常组织相比,RP1-261G23.7在胶质瘤组织中表达上调(P<0.01)。与人脑星型胶质正常细胞系(HEB)相比,RP1-261G23.7在四种胶质瘤细胞系中表达均上调(P<0.01),U87MG细胞中RP1-261G23.7相对表达量最高(P<0.01)。与si-NC组相比,敲减RP1-261G23.7显著抑制了U87MG细胞的增殖(P<0.05)和划痕愈合(P<0.01)。RP1-261G23.7能够直接互补结合miR-525-5p(P<0.01)。与si-NC组相比,敲减RP1-261G23.7表达显著促进了U87MG细胞中miR-525-5p的表达(P<0.01),NF-κB信号通路蛋白表达显著下降(P<0.01)。结论:胶质瘤组织和细胞系中RP1-261G23.7表达明显上调,敲减RP1-261G23.7通过促进miR-525-5p表达、干扰NF-κB信号通路活化,抑制胶质瘤U87MG细胞的增殖和迁移,可能为胶质瘤的靶向治疗开辟新的路径。     

RNASE4异常表达对胶质瘤细胞侵袭迁移的影响及机制研究

摘 要：［目的］ 阐明RNASE4在胶质瘤细胞迁移和侵袭中的潜在作用及机制。［方法］ 利用实时定量PCR（qPCR）检测RNASE4在正常胶质细胞和胶质瘤细胞系中的表达水平。将胶质瘤U87细胞分为对照组、siRNA-NC组和siRNA-RNASE4组。在体外细胞中瞬时转染siRNA-RNASE4，利用CCK-8法检测胶质瘤细胞U87增殖率。采用伤口愈合实验和Transwell小室实验检测U87细胞的侵袭和迁移能力。通过定量PCR和Western blot实验检测各组细胞中相关因子的表达。［结果］ RNASE4在胶质瘤细胞系T98（1.34±0.06，P<0.01）、A172（1.79±0.10，P<0.01）、U251（1.86±0.09，P<0.01）、U118（2.19±0.17，P<0.01）和U87（2.64±0.17，P<0.01）中较NHAs细胞系均异常高表达（1.00±0.05）。在胶质瘤细胞U87中下调RNASE4表达引起细胞增殖活力下降（48h，P<0.05；72h，P<0.01）。同时，siRNA-RNASE4组U87细胞的划痕愈合能力（46.0±4.6 vs 87.2±3.6，P<0.01）以及细胞侵袭能力（72.5±4.7 vs 147.8±16.8，P<0.01）均明显降低。下调RNASE4表达抑制了BCL2、MMP9基因表达，并促进各组细胞中E-cadherin表达（P<0.01）。［结论］ RNASE4能够通过调节BCL2、MMP9等因子的表达来调节胶质瘤细胞的增殖与迁移能力，发挥促癌因子的作用。     

RNA干扰介导EphB4基因表达下调对胶质瘤细胞系U251生长的影响

目的：探讨靶向EphB4基因的小干扰RNA（small interference RNA，siRNA）对胶质瘤细胞EphB4 mRNA表达及细胞生长的影响。方法：体外化学合成针对人EphB4出基因的小干扰RNA并转染恶性胶质瘤U251细胞系后，RT-PCR法检测EphB4 mRNA表达的变化，CCK-8检测法观察RNA干扰对肿瘤细胞增殖活性的影响，FCM检测细胞周期变化，划痕实验观察细胞的迁移能力，采用Transwell小室穿膜细胞的数量来反应细胞的侵袭能力。结果：U251细胞转染siRNA-EphB4 100nmol／L后，EphB4 mRNA的表达水平下降了75．0％，细胞增殖抑制表现为剂量依赖性，FCM检测与阴性对照相比，细胞周期不同程度阻滞于亚G。期；并且细胞的迁移能力与对照组比较有所下降，Transwell小室穿膜细胞与对照组比较明显减少。结论：siRNA—EphB4能够明显靶向并抑制U251细胞中EphB4基因的表达，而EphB4基因表达下调可使U251细胞增殖受到影响，细胞周期出现凋亡峰。转染siRNA-EphB4后U251细胞的迁移和侵袭能力受到不同程度的抑制。提示抑制EphB4基因表达有可能成为胶质瘤治疗的新方法。     

Stathmin蛋白的研究进展

目的: 探讨趋化因子受体CXCR4表达水平对人肺癌细胞转移潜能的影响.方法: 采用RT-PCR,FACS检测趋化因子受体CXCR4在肺癌细胞株95C,95D细胞的表达.构建CXCR4正义和反义真核表达质粒,用脂质体法转染至95C和95D细胞内,通过G418筛选出稳定转染株.通过趋化和趋化侵袭实验测定转染前后细胞对基质衍生因子(SDF-1α)的迁移能力,通过明胶酶谱法检测MMP-2活性,通过FACS检测细胞对血管内皮细胞的黏附能力.结果: CXCR4正义和反义真核表达质粒pcDNA3-X4和pcDNA3-ASX4能明显上调或下调肺癌细胞表面CXCR4的表达,上调95C细胞表面CXCR4的表达可以增强其对SDF-1α的趋化反应性、MMP-2活性及其对血管内皮细胞的黏附能力.相反,下调95D细胞表面CXCR4的表达抑制了其对SDF-1α的趋化反应性、MMP-2活性及其对血管内皮细胞的黏附能力.结论: 上调或下调肺癌细胞表面CXCR4表达可以显著增强或抑制其转移潜能,提示CXCR4表达水平与肺癌细胞转移潜能有关.     

Free radicals as carcinogens and their quenchers as anticarcinogens

An oxygen dependent signal was detected, late in the 1950s by electron spin resonance (ESR) in a saline solution of hematoporphyrin (Hp) excited by light. This signal expressed a free radical consisting of ‘some kind of an association between Hp and oxygen’, that Smalleret al. called ‘oxyradical’ (HpOO). It soon opened a new level of understanding in carcinogenesis triggered by photodynamic substances, including Hp itself, polycyclic hydrocarbons (PCHs), as well as any carcinogen involving molecular species activated by radiation and/or metabolic reaction. Early in the 1960s, this prompted the discovery of benzo(a)pyrene (BP) photocarcinogenic enhancement (BP-PCE) in mice, probably due to an increase in free oxygen radical generation following correct light exposure. This assumption was confirmed in 1980 by the fact that mice orally loaded with antioxidants and radical quenchers, such as beta-carotene (BC) and cantaxanthin (CX), were protected against BP-PCE at 100% and against total BP carcinogenicity at more than 60%. These achievements were presented as the bases of the current explosion of interest in biology and medicine in building up the new field of chemoprevention against cancer and other chronic diseases by supplementation with antioxidant vitamins, retinoids and especially carotenoids and their synergistic association. The relevant findings of this research obtained in the last decade inin vitro andin vivo experiments as well as human interventions are reported and discussed with personal contributions. This report is to honor the memory of Douglas E. Smith, a pioneer in the prevention of ionizing radiation damage, and Bernard Smaller, a gifted developer of the ESR in basic science. Both were prominent scientists and friends of the authors. The first part of this report relates to work carried together in the Argonne National Laboratory, Argonne, Illinois, U.S.A. 1959.     

Mitogens as motogens

Numerous in vivo methodologies have documented the invasivebehavior of glioma cells through normal brain parenchyma.Glioma cell locomotion has also been assessed witha number of in vitro assays including theBoyden chamber and other chemotaxis assays, colloidal goldcell tracking, analysis of migration of cells tumorcells from spheroids, confrontation cultures of glioma cellswith aggregates of non-neoplastic tissue, time-lapse video microscopy,electron microscopic examination of the cytomorphologic correlates ofcell motility, the radial dish assay, and quantitativeenzyme immunoassay of proteins associated with invasion (e.g.laminin). Several of these techniques have been specificallymodified to assess the effects of cytokines onglioma cell motility in vitro. Cytokines studied utilizingthese methods include: epidermal growth factor (EGF), basicfibroblast growth factor (bFGF), the bb dimer ofplatelet-derived growth factor (PDGFbb), nerve growth factor (NGF),interleukin 2 (IL-2), transforming growth factors alpha andbeta 1 (TGF and TGFstraat₁), and tumor necrosisfactor alpha (TNF). This review summarizes the investigationalmethods used to evaluate random and directional gliomacell motility and invasion in vivo and invitro. The roles of specific mitogens as motogens,as evaluated with these methods are then presented.     

Free radicals as carcinogens and their quenchers as anticarcinogens.

An oxygen dependent signal was detected, late in the 1950s by electron spin resonance (ESR) in a saline solution of hematoporphyrin (Hp) excited by light. This signal expressed a free radical consisting of 'some kind of an association between Hp and oxygen', that Smaller et al. called 'oxyradical' (HpOO.). It soon opened a new level of understanding in carcinogenesis triggered by photodynamic substances, including Hp itself, polycyclic hydrocarbons (PCHs), as well as any carcinogen involving molecular species activated by radiation and/or metabolic reaction. Early in the 1960s, this prompted the discovery of benzo(a)pyrene (BP) photocarcinogenic enhancement (BP-PCE) in mice, probably due to an increase in free oxygen radical generation following correct light exposure. This assumption was confirmed in 1980 by the fact that mice orally loaded with antioxidants and radical quenchers, such as beta-carotene (BC) and cantaxanthin (CX), were protected against BP-PCE at 100% and against total BP carcinogenicity at more than 60%. These achievements were presented as the bases of the current explosion of interest in biology and medicine in building up the new field of chemoprevention against cancer and other chronic diseases by supplementation with antioxidant vitamins, retinoids and especially carotenoids and their synergistic association. The relevant findings of this research obtained in the last decade in in vitro and in vivo experiments as well as human interventions are reported and discussed with personal contributions.     

Patients as partners

Why should patients be partners in their treatment and care? How has the concept developed? How can this relationship be achieved? What benefits does it produce? This article considers the various factors that have influenced public and professional attitudes in recent years, creating a climate where patients feel they may express a wish to participate in treatment and care, and in which health-care professionals accept and encourage this. Having reached common ground in terms of attitude, how can this partnership be achieved in practice? What is required of both parties to reach a desired outcome? Meeting the informational needs of patients, relatives and friends is only one aspect of this process; others are also discussed. Finally, what are the benefits, real or imaginary, to patient and professional - financial, and in terms of research? The evidence, and in some cases the lack of it, is presented.