脑胶质瘤分子病因相关组织芯片CyclinB1表达及其临床意义的研究

目的:探讨CyclinB1在针对胶质瘤发生、发展相关组织芯片中的表达及意义.方法:构建针对人脑胶质瘤发生、发展分子机制研究的组织芯片,应用免疫组化EnVision法检测CyclinB1在组织芯片标本中的表达,分析其表达率及表达强度.结果:CyclinB1染色阳性表达主要位于细胞核.在71例人脑胶质瘤组织中,CyclinB1阳性表达率为52.1%,与正常成人脑组织(18.2%)比较,差异有统计学意义(x2=5.15,P<0.05).Ⅰ、Ⅱ、Ⅲ和Ⅳ级人脑胶质瘤组织CyclinB1阳性表达率分别为11.2%、44.0%、65.2%和71.4%;病理级别与CyclinB1的阳性表达率呈正相关(r=0.959,P<0.05).两种胶质瘤细胞系多细胞球体SHG44细胞和GBM细胞的CyclinB1阳性表达率高于神经干细胞、脑肿瘤干细胞(x2=9.60,P<0.01);人胚胎脑组织、裸鼠移植瘤和裸鼠骨髓中也呈过表达.结论:CyclinB1的表达强度能客观反映被测细胞的增殖活性,处于非增殖状态的干细胞低表达或不表达.它的高表达与肿瘤恶性程度相一致.     

ABCG2在胶质瘤组织中的表达及意义

背景与目的:三磷酸腺苷结合盒转运体成员ABCG2(ATP-binding cassette superfamily G member 2)是源于造血干细胞的标志物之一,其在神经胶质瘤发生发展相关组织和细胞中的表达情况还不清楚.本研究检测ABCG2在不同恶性程度人脑胶质瘤组织标本、裸小鼠移植瘤标本、体外细胞系球体和胶质瘤干细胞球体中的表达情况并分析其意义.方法:制作布有不同恶性程度人脑胶质瘤组织标本、裸小鼠移植瘤标本、体外细胞系球体和胶质瘤干细胞球体等的组织芯片,用免疫组化方法检测ABCG2在组织芯片中的表达情况.结果:在71例人脑胶质瘤组织标本中ABCG2的阳性率为26.8%,其中Ⅰ级11.1%,Ⅱ级8.0%,Ⅲ级43.5%,Ⅳ级42.9%;Ⅰ～Ⅱ级与Ⅲ～Ⅳ级相比差异具有统计学意义(X2=10.710,P=0.001).在神经干细胞、裸小鼠移植瘤、胶质瘤干细胞球体表达率为100%.在多种正常组织中亦有不同程度的表达.在胶质瘤临床标本中ABCG2阳性细胞呈亲血管分布.结论:ABCG2在胶质瘤干细胞、恶性程度高的胶质瘤组织标本和移植瘤组织中高表达,并且呈亲血管分布.     

COX-2、Ki-67与nm23在人脑胶质瘤组织中的表达及临床意义

目的研究COX-2、Ki-67及nm23在人脑胶质瘤中的表达及其临床意义。方法应用免疫组织化学S-P法检测10例正常脑组织和56例人脑胶质瘤组织巾COX-2、Ki-67及nm23的表达。结果COX-2和Ki-67在正常脑组织中均不表达,而在各病理级别的人脑胶质瘤组织中均有表达,各组间差异有统计学意义（P〈0．05）,COX-2和Ki-67的表达强度随脑胶质瘤病理级别呈递增趋势,且COX-2和Ki-6的表达呈显著正相关（r=0．567,P〈0．05）;nm23在正常脑组织和各病理级别人脑胶质瘤组织中均有表达,各组问差异有统计学意义（P〈0．05）,且Ki-6和nm23的表达呈显著负相关（r=-0．388,P〈0．05）。结论COX-2和Ki-67的表达强度随胶质瘤恶性程度的增加而增加;nm23的表达强度随胶质瘤恶性程度的增加而减少;COX-2、Ki-67及nm23与人脑胶质瘤的发生和发展有关系,三者可作为胶质瘤恶性程度评估的重要生物学参考指标。     

CD133和巢蛋白阳性细胞在脑胶质瘤中的表达及分布特征

目的：探讨脑肿瘤干细胞标记物CD133、巢蛋白（Nestin）和CD34标记的微血管密度（CD34-MVD）在人脑胶质瘤中的表达情况,分析三者的关系及其意义。方法：应用免疫组化方法分析62例人脑胶质瘤中的CD133、Nestin和CD34-MVD的表达情况。结果：脑肿瘤干细胞标记物CD133、Nestin在正常脑组织中未见表达,在低级别脑胶质瘤中低表达,在高级别脑胶质瘤中高表达,各组之间表达有显著性差异（P〈0．05）。CD133表达与Nestin表达之间有相关性,两者呈正相关（P〈0．05）。CD133、Nestin均阳性染色组的CD34-MVD值高于两者均阴性组（P〈0．01）。结论：CD133和Nestin表达呈正相关性,两者与人脑胶质瘤的病理分级呈正相关,与CD34-MVD在分布及数量上存在相关性。     

量子点免疫荧光技术在脑胶质瘤组织芯片上的应用

目的探讨窖蛋白．1（caveolin．1,Cav-1）、细胞外基质金属蛋白酶诱导因子（CD147）在人脑胶质瘤中的表达以及临床意义,并分析二者的相关性。方法在脑胶质瘤组织芯片上,利用新型半导体纳米微晶体——量子点（QDs）免疫荧光技术检测Cav-1、CD147的蛋白表达。结果Cav-1和CD147蛋白在人脑胶质瘤组织中的阳性表达率分别为42．86％、71．43％,与正常脑组织相比,CD147蛋白的表达差异有显著性（P=0．042）。Cav-1和CD147蛋白表达与胶质瘤患者的性别、年龄和组织学类型均无关（P〉0．05）,而与病理学分级均显著相关（P〈0．05）。Cav-1和CD147蛋白在人脑胶质瘤组织中的阳性表达呈显著负相关（P=0．001,r=-0．730）。结论量子点免疫荧光技术能精确检测人脑胶质瘤组织芯片上不同蛋白的定位。Cav-1和CD147蛋白在人脑胶质瘤的恶性进展中可能起拮抗作用。     

人脑胶质瘤中EGFR和p27~(kip1)表达及其意义

[目的]研究表皮生长因子受体（EGFR）和p27^kip1在人脑胶质瘤中的表达及意义。[方法]采用免疫组化SP法检测60例人脑胶质瘤中EGFR、p27^kip1的表达,分析两者相关性及其与人脑胶质瘤病理分级的关系。[结果]EGFR在Ⅱ级、Ⅲ级、Ⅳ级人脑胶质瘤中阳性表达率分别为52．38％、60．87％和87．50％,随人脑胶质瘤级别增加EGFR表达率升高（P〈0．05）;p27^kip1在Ⅱ级、Ⅲ级、Ⅳ级人脑胶质瘤中阳性表达率分别为57．14％、26．09％和12．50％。随人脑胶质瘤级别增加p27^kip1表达率下降（P〈0．05）;两者表达呈负相关（rs=-0．616,P〈0．05）。[结论]EGFR表达强度随人脑胶质瘤级别的升高而增加,而p27^kip1表达随人脑胶质瘤级别的升高而降低,两者在胶质瘤的浸润、迁移和肿瘤抑制中可能有相互凋节作用。     

CD133、Nestin在星形细胞肿瘤中的表达及与病理级别的关系

背景与目的：脑胶质瘤治疗效果一直不理想，这与脑胶质瘤中存在一部分肿瘤干细胞有关。本研究旨在探讨脑肿瘤干细胞标记物CDl33及Nestin在脑星形细胞肿瘤中的表达的临床意义。方法：采用免疫组织化学SP法检测CDl33、Nestin在127例脑星形细胞肿瘤组织中的表达。结果：（1）按照WH02000年的神经系统肿瘤分类分级标准所有标本分为I级15例，Ⅱ级42例，Ⅲ级42例，Ⅳ级28例。（2）脑肿瘤干细胞标记物在低级别星形细胞肿瘤中低表达，在高级别星形细胞肿瘤中高表达，在低级别和高级别之间表达有显著性差异（P=0．000）。（3）CDl33表达与Nestin表达之间有相关性，二者呈正相关（r=0．411，P=0．000）。（4）CDl33阳细胞性率与患者性别、年龄无关（P=0、136、P=0．412），Nestin的阳性细胞率与患者性别、年龄无关（P=0．412、P=0．153）。结论：CDl33及Nestin在低级别星形细胞瘤中低表达．在高级别星形细胞肿瘤中高表达。     

CDl33、Nestin在星形细胞肿瘤中的表达及与病理级别的关系

背景与目的：脑胶质瘤治疗效果一直不理想，这与脑胶质瘤中存在一部分肿瘤干细胞有关。本研究旨在探讨脑肿瘤干细胞标记物CDl33及Nestin在脑星形细胞肿瘤中的表达的临床意义。方法：采用免疫组织化学SP法检测CDl33、Nestin在127例脑星形细胞肿瘤组织中的表达。结果：（1）按照WH02000年的神经系统肿瘤分类分级标准所有标本分为I级15例，Ⅱ级42例，Ⅲ级42例，Ⅳ级28例。（2）脑肿瘤干细胞标记物在低级别星形细胞肿瘤中低表达，在高级别星形细胞肿瘤中高表达，在低级别和高级别之间表达有显著性差异（P=0．000）。（3）CDl33表达与Nestin表达之间有相关性，二者呈正相关（r=0．411，P=0．000）。（4）CDl33阳细胞性率与患者性别、年龄无关（P=0、136、P=0．412），Nestin的阳性细胞率与患者性别、年龄无关（P=0．412、P=0．153）。结论：CDl33及Nestin在低级别星形细胞瘤中低表达．在高级别星形细胞肿瘤中高表达。     

周期蛋白依赖性激酶1在胶质瘤组织中的表达及其沉默对胶质瘤细胞恶性表型的影响

目的探讨周期蛋白依赖性激酶1(CDK1)在胶质瘤组织中的表达及其沉默对胶质瘤细胞恶性表型的影响。方法利用自建的人脑胶质瘤体外细胞系SHG44及其移植瘤组织、脑肿瘤干细胞、神经干细胞、不同恶性程度的胶质瘤手术标本构建组织芯片,免疫组化染色检测其CDK1蛋白表达;应用RNA干扰技术使CDK1在SHG44细胞及其移植瘤组织中沉默,观察其随后发生的表型变化。结果CDK1在临床标本中随胶质瘤恶性程度升高表达强度增加,Ⅰ-Ⅳ级的阳性表达率分别为22.2%、40.0%、69.6%和78.6%(P=0.01)。体外培养的人脑胶质瘤SHG44细胞球体和裸小鼠皮下移植瘤CDK1表达强度均较高,而神经干细胞和脑肿瘤干细胞球体表达强度低,在细胞增殖活性高的人胚胎脑组织和裸小鼠骨髓中CDK1亦高表达,但在正常成人脑组织低表达。C1和C3体外转染SHG44细胞后,将细胞周期阻滞在G2/M期,而且诱导了凋亡,细胞凋亡率分别上升至27.8%和36.5%。CDK1沉默的裸鼠皮下移植瘤瘤重明显降低,细胞凋亡率为57.1%,明显高于对照组(8.5%)。结论CDK1的高表达呵促进胶质瘤的发生和发展,CDK1沉默后的肿瘤细胞恶性表型可得到控制,CDK1可作为胶质瘤病因分子行进一步研究。     

人脑胶质瘤中凋亡相关基因survivin和p53的表达研究

目的探讨凋亡相关基因survivin和p53在人脑胶质瘤发生发展中的作用及其相互关系。方法采用免疫组化S-P法检测10例正常脑组织及75例脑胶质瘤标本中survivin、p53的表达情况并分析二者之间的关系。结果survivin在正常对照组和胶质瘤组中的阳性表达率为0％和45．3％，差异有显著性（P〈0．05）。胶质瘤Ⅰ级、Ⅱ级和Ⅲ，Ⅳ级，survivin的阳性表达率分别为15．0％、33．3％、74．2％，差异有极显著性（P〈0．001）。p53在正常脑组织和人脑胶质瘤组织中阳性表达率为0％和41、3％，差异有显著性（P〈0．05）。胶质瘤Ⅰ级、Ⅱ级和Ⅲ-Ⅳ级，p53的阳性表达率0％、41．7％、67．7％，差异有极显著性（P〈0．001）。75例胶质瘤组织中survivin与p53共阳性23例，共阴性33例，二者的表达呈正相关（rs=0．438，P〈0．01）。结论1）survivin的阳性表达率随胶质瘤恶性程度而升高；2）突变型p53蛋白的活性表达与肿瘤的组织病理类型、分化程度有关，p53基因的突变参与脑胶质瘤的发生发展；3）p53的突变与survivin的激活在胶质瘤的发生发展中可能起协同作用。     

Decreased cyclin B1 expression contributes to G2 delay in human brain tumor cells after treatment with camptothecin

DNA damage produces delayed mitosis (G2/M delay) in proliferating cells, and shortening the delay sensitizes human malignant glioma and medulloblastoma cells to cytotoxic chemotherapy. Although activation of the cyclin-dependent kinase CDC2 mediates G2/M transition in all tumor cells studied to date, regulation of CDC2 varies between tumor types. Persistent hyperphosphorylation of kinase and reduced cyclin expression have been implicated as mediators of treatment-induced G2 delay in different tumor models. To evaluate regulation of G2/M transition in human brain tumors, we studied the expression and/or activity of CDC2 kinase and cyclins A and B1 in U-251 MG and DAOY medulloblastoma cells after their treatment with camptothecin (CPT). Synchronized cells were treated during S phase, then harvested at predetermined intervals for evaluation of cell cycle kinetics, kinase activity mRNA, and protein expression. CPT produced G2 delay associated with decreased CDC2 kinase activity and cyclin B1 expression. Kinase activity was associated with CDC2 bound to cyclin B1, not cyclin A, in both cell lines. Cyclin A mRNA and protein expression were reduced after CPT treatment; however, decreased protein expression was short lived and moderate in the glioma and primitive neuroectodermal tumor/medulloblastoma cells, respectively. We conclude that G2 delay is a common response of brain tumor cells to chemotherapy with topoisomerase I inhibitors and that a mechanism of this delay may be reduced expression of cyclin B1.     

Molecular Study on Differentiation-Associated Genes Involved in Both Malignant Progression of Glioma and Differentiation of Human Fetal Neural Stem Cells

I n the early 1990s, Reynolds and Weiss provided the initial evi- dence that multipotent neural stem cells of the adult rodent brain can be identified and isolated in vitro following mitogen stimulation.[1] There is no doubt that neural stem cells (NSCs) …     

Functional analysis of the DEPDC1 oncoantigen in malignant glioma and brain tumor initiating cells

DEP domain containing 1 (DEPDC1) is a novel oncoantigen expressed in cancer cells, which presents oncogenic activity and high immunogenicity. Although DEPDC1 has been predicted to be a useful antigen for the development of a cancer vaccine, its pathophysiological roles in glioma have not been investigated. Here, we analyzed the expression and function of DEPDC1 in malignant glioma. DEPDC1 expression in glioma cell lines, glioma tissues, and brain tumor initiating cells (BTICs) was assessed by western blot and quantitative polymerase chain reaction (PCR). The effect of DEPDC1 downregulation on cell growth and nuclear factor kappa B (NFκB) signaling in glioma cells was investigated. Overall survival was assessed in mouse glioma models using human glioma cells and induced mouse brain tumor stem cells (imBTSCs) to determine the effect of DEPDC1 suppression in vivo. DEPDC1 expression was increased in glioma cell lines, tissues, and BTICs. Suppression of endogenous DEPDC1 expression by small interfering RNA (siRNA) inhibited glioma cell viability and induced apoptosis through NFκB signaling. In mouse glioma models using human glioma cells and imBTSCs, downregulation of DEPDC1 expression prolonged overall survival. These results suggest that DEPDC1 represents a target molecule for the treatment of glioma.     

miR-29a对胶质瘤细胞CDC42表达及迁移和侵袭的影响

  目的  探讨胶质瘤miR-29a与CDC42表达的相互关系及其对肿瘤细胞侵袭迁移的影响。  方法  采用组织微阵列及锁定寡核苷酸原位杂交技术, 检测60例不同级别胶质瘤及10例非肿瘤对照脑组织中miR-29a的表达水平, 采用qRT-PCR、Westernblot及Transwell培养, 分别检测U251及其稳定表达miR-29a和无义对照序列的亚细胞系(U251-miR-29a及U251-scr)miR-29a、CDC42 mRNA及蛋白的表达及其迁移和侵袭能力, 并分析其相互关系。  结果  各级别胶质瘤miR-29a表达水平均显著低于对照脑组织, 并随肿瘤级别升高而减少, 差异均有统计学意义(P < 0.01)。U251-miR-29a的miR-29a表达量明显高于U251和U251-scr(P < 0.001), 其CDC42 mRNA和蛋白表达量明显降低(P < 0.05);CDC42 mRNA与其蛋白表达量呈正相关(r=0.834, P < 0.01), 而与miR-29a表达量呈负相关(r=-0.979, P < 0.01)。U251-miR-29a的迁移和侵袭能力明显低于U251和U251-scr(P < 0.001), 并均与CDC42蛋白表达量呈正相关(r=0.828, P < 0.01)。  结论  miR-29a表达水平是评价胶质瘤良恶性的重要参考指标, 胶质瘤细胞miR-29a表达异常减少是CDC42上调及迁移侵袭能力增强的重要因素, 补充外源性miR-29a可抑制靶基因CDC42表达, 阻止其侵袭迁移, 提示恶性胶质瘤基因治疗中具有重要的潜在应用价值。      

The progression of gliomas is associated with cancer stem cell phenotype

Since cancer stem cells in brain tumors were introduced, there have been few explanations regarding the role of cancer stem cells in the progression of glioma. Here, we investigated their major molecular changes in tumor progression in relation to the stem cell subpopulation. Using 12 surgical specimens of gliomatosis cerebri (GC) in the early and advanced stages, we measured the expression of a panel of cell proliferation, microvessel density, microvessel areas, angiogenic factors and their associated receptors. In addition, expression of neural stem cell markers and associated cytokines were examined in tumor tissues by quantitative real-time RT-PCR. Comparing the biological characteristics between the initial infiltrating lesions (n=7) and progressed lesions (n=5), Sox2 and Musashi-1 were expressed in the tumor tissue at an early and a progressed state. Contrary to the early infiltrative phase representing angiogenesis-independent growth, GC with progression showed that nestin (+), PCNA (+) cells and total vessel area (angioectasia) were markedly increased with a higher expression of proangiogenic molecules and their receptors. These results suggest that tumor progression is mediated by cancer stem cells and cross-talk of cancer stem cells along with their environment and are closely associated with angiogenesis-dependent progression and -independent growth.     

MSP58基因表达水平与人脑胶质瘤恶性度的关系

目的:观察MSP58基因表达水平与人脑胶质瘤恶性度的关系.方法:在41例按WHO分类和分级标准为Ⅰ-Ⅳ级的人脑胶质瘤标本、4株人脑胶质瘤细胞系(U251,U87,BT325和SHG44)以及6例正常脑组织标本中,运用半定量RT - PCR及蛋白质印迹法检测MSP58 mRNA和蛋白的表达水平.结果:MSP58 mR-NA和蛋白在人脑胶质瘤组织中存在表达,其表达水平随人脑胶质瘤恶性度的增加而升高.在正常脑组织和恶性脑胶质瘤之间,以及脑胶质瘤Ⅰ-Ⅳ级病理级别间比较均有显著差异.MSP58 mRNA和蛋白在U251、U87、BT325和SHG44细胞中均有高表达.结论:MSP58 mRNA和蛋白在人脑胶质瘤中高表达,其表达水平与脑胶质瘤的恶性度有关,提示MSP58在脑胶质瘤的形成和恶性演进中具有重要作用.     

Downregulation of laminin alpha4 chain expression inhibits glioma invasion in vitro and in vivo

The laminin family is a structural constituent of the extracellular matrix that plays an essential role in promoting the motility of infiltrative tumor cells. We investigated the role of laminin alpha4 chain, a subset of laminin-8, -9 and -14, in the motile and invasive activities of human glioma cells. All malignant glioma cell lines examined expressed more mRNA for the laminin alpha4 and beta1 chains than for the beta2 chain, indicating that these cells predominantly express the laminin-8 isoform. Introducing an antisense oligonucleotide for laminin alpha4 chain (AS-Ln-alpha4) into the glioma cells resulted in downregulation of laminin alpha4 expression. AS-Ln-alpha4 also significantly suppressed glioma cell adhesion and migration. Furthermore, invasiveness was significantly reduced in cells transfected with AS-Ln-alpha4 compared to those transfected with the sense oligonucleotide (S-Ln-alpha4). Indeed, when glioma spheroids were implanted into rat brain slices, AS-Ln-alpha4-transfected cells failed to invade surrounding normal brain tissues. In addition, intracerebral injection of glioma cells transfected with AS-Ln-alpha4 into nude mice resulted in the formation of a noninvasive tumor, whereas injection of cells transfected with S-Ln-alpha4 resulted in diffuse invasion of brain tissue. These results suggest that mainly laminin-8 is essential for the invasive activity of human glioma cells; thus, a novel therapeutic strategy could target this molecule to treat patients with malignant glioma.     

4F2hc表达与人脑胶质瘤病理级别增殖和血管形成的关系

  目的  探讨细胞表面抗原4F2重链(4F2 heavy chain, 4F2hc)在人脑胶质瘤组织中的表达水平及其与胶质瘤病理学特征、细胞增殖以及血管形成的关系。  方法  采用免疫组化方法检测4F2hc、Ki-67和CD34在62例人脑胶质瘤组织中的表达, 计数Ki-67标记指数(Ki-67 LI)和微血管密度(MVD)。  结果  4F2hc在胶质瘤中高表达, 其免疫阳性染色既定位于瘤细胞也定位于血管内皮; 4F2hc表达随胶质瘤病理级别升高而明显增强(P=0.001), 在高度恶性胶质瘤中4F2hc表达明显强于低度恶性胶质瘤(P=0.002);4F2hc表达与胶质瘤Ki-67标记指数存在明显正相关(P=0.003), 但与微血管密度无明显相关性(P=0.214)。  结论  4F2hc与胶质瘤的发生和发展关系密切, 可能在胶质瘤的恶性增殖过程中具有重要作用。