脑胶质瘤Th1/Th2类细胞因子的漂移及意义

我们采用逆转录聚合酶链反应(ＲＴ ＰＣＲ)方法 ,检测了 4株脑胶质瘤细胞株、5 2例新鲜脑胶质瘤标本和 15例脑胶质瘤患者外周血标本的Ｔｈ1/Ｔｈ2类细胞因子的表达情况 ,初步探讨了脑胶质瘤Ｔｈ2类细胞因子漂移的意义。1.材料与方法 :胶质瘤细胞株ＳＨＧ 44、Ｕ2 5 1、Ｃ6和 9Ｌ ,购自上海细胞生物研究所或本室保存。胶质瘤组织标本5 2例 ,取自我院患者 ,术前均未接受放疗、化疗或免疫治疗。术后病理 :星形细胞瘤Ⅰ级 4例 ,星形细胞瘤Ⅱ级 6例 ,间变性星形细胞瘤 2 3例 ,多形性胶质母细胞瘤 9例 ,少突胶质细胞瘤 3例 ,脉络丛乳头状瘤 1例 ,…     

血管内皮生长因子在脑胶质瘤中自分泌研究

目的 观察血管内皮生长因子(VEGF)在脑胶质瘤中自分泌现象.方法 采用免疫组织化学及图像分析方法 ,检测74例脑胶质瘤中肿瘤细胞上血管内皮生长因子受体VEGFR1和VEGFR2的表达,取其中42例检测VEGF的表达的情况.结果 VEGF在胶质瘤中表达强度与肿瘤病理级别明显相关(P＜0.05,r=0.331);VEGFR1、2在胶质瘤瘤细胞中表达强度吸光值分别为:正常组织(0.2104±0.0297)和(0.2100±0.0257);WHO I(0.2328±0.0194)和(0.2324±0.0186);WHOⅡ级(0.2533±0.0155)和(0.2514±0.0147);WHO Ⅲ级(0.2709±0.0203)和(0.2720±0.0149);WHOⅣ级(0.2906±0.0282)和(0.2834±0.0285);表达强度与胶质瘤病理级别明显相关(P＜0.01,r=0.748;P＜0.01,r=0.754).结论 在脑胶质瘤中存在VEGF的旁分泌和自分泌现象,并与病理级别有关.     

血管内皮生长因子在脑胶质瘤中自分泌现象对增殖的影响

FR2呈强表达同时Ki-67呈弱表达1例,VEGFR2呈弱表达同时Ki-67呈强表达6例,经一致性分析Kappa=0.543,P＜0.01,两者的共表达存在一致性.结论 脑胶质瘤瘤细胞可能通过VEGF自分泌现象介导瘤细胞的增殖,尤以VEGFR2参与介导的可能性大.     

TRAIL受体在人骨肉瘤组织中的表达

目的 研究肿瘤坏死因子相关凋亡诱导配体(TRAIL)受体在骨肉瘤细胞中的表达情况。方法 应用逆转录—聚合酶链反应(RT—PCR)对22例骨肉瘤组织标本、MG—63骨肉瘤细胞株、U251脑胶质瘤细胞株以及正常人外周血淋巴细胞TRAILR1—R4 mRNA表达进行检测。结果 22例骨肉瘤组织标本中15例同时表达TRAILR1、-R2和-R3，4例同时表达TRAIL-R1和-R2，只表达TRAIL-R1或-R2者3例，所有标本中均未检测到TRAIL，-R4表达；MG-63骨肉瘤细胞株、U251脑胶质瘤细胞株以及正常人外周血淋巴细胞则均检测到TRAILR1、-R2和-R3的联合表达。结论 死亡受体TRAIL-R1，R2在骨肉瘤中的普遍表达，是TRAIL诱导骨肉瘤细胞凋亡的分子基础；死亡受体和诱骗受体的差异性分布，并非TRAIL对选择性杀伤肿瘤细胞的关键性因素，可能还受其它因子调控。     

膀胱癌组织中Wnt拮抗因子分泌型卷曲相关蛋白1甲基化和异常表达的意义

目的 探讨Wnt拮抗因子分泌型卷曲相关蛋白1(SFRP1)甲基化和表达在膀胱癌发病机制中的作用. 方法 采用甲基化特异性聚合酶链反应检测人膀胱癌细胞株T24、5637和SCaBER及45例膀胱癌组织和对应癌旁正常组织的SFRP1基因甲基化状态,蛋白质印迹方法检测SFRP1蛋白表达. 结果 SFRP1在膀胱癌细胞株T24和5637中呈现甲基化状态,而SCaBER细胞未检出SFRP1甲基化.将去甲基化试剂5’-氮杂-脱氧胞苷酸(1μg/ml)加入发生甲基化的膀胱癌细胞株T24和5637后,细胞SFRP1 mRNA和蛋白出现表达.45例膀胱癌组织中SFRP1甲基化表型28例(62.2％),癌旁正常组织6例(13.3％),组间频率差异有统计学意义(P＜0.01). 结论 SFRP1甲基化及表达下调可能参与了膀胱癌的发病过程.     

肝癌组织辅助性T细胞-2细胞因子强势分泌研究

目的　探讨人肝癌组织中辅助性T细胞亚群 (Th1/Th2 )细胞因子表达模式。方法　收集我科手术切除肝癌组织标本 15例 ,肝良性疾病或肝硬化门静脉高压手术患者肝组织标本 15例作为对照 ,β 肌动蛋白 (β actin)作为参照 ,采用半定量逆转录 聚合酶联反应 (RT PCR)方法检测人肝癌组织中γ 干扰素 (IFN γ)和白细胞介素 4(IL 4)mRNA的表达。结果　人肝癌组织中IFN γmRNA表达 (相对数 :1.18± 0 .3 1)比对照组肝组织中IFN γmRNA表达弱 (相对数 :1.86±0 .5 0 ) ,而IL 4mRNA表达 (相对数 :1.73± 0 .5 1)比对照组强 (相对数 :0 .94± 0 .41,P <0 .0 5 )。结论　人肝癌组织中Th1类细胞因子表达弱 ,Th2类细胞因子表达强 ,呈现Th2细胞因子表达模式 ,肝癌组织局部微环境处于免疫抑制状态 ,肿瘤细胞易发生免疫逃逸     

RAC3在脑胶质瘤组织的表达及其对脑胶质瘤细胞迁移与侵袭能力的影响

目的探索ras相关C3肉毒杆菌毒素底物3(ras-related C3 botulinum toxin substrate 3, RAC3)在脑胶质瘤组织的表达情况, 及其对脑胶质瘤细胞迁移与侵袭能力的影响。方法用免疫组化实验检测商丘市第一人民医院神经外科收治的57例脑胶质瘤患者组织及其癌旁组织标本中RAC3的表达情况。将脑胶质瘤细胞U87MG分为实验组和对照组, 实验组U87MG细胞转染RAC3-siRNA质粒, 对照组U87MG细胞转染MOCK-siRNA质粒;荧光定量PCR检测各组细胞RAC3 mRNA含量, 蛋白质免疫印迹法检测各组细胞RAC3、MMP2的表达情况;Transwell检测各组细胞的迁移与侵袭能力。结果脑胶质瘤患者组织RAC3阳性率为89.47%(51/57), 癌旁组织中表达率为14.04%(8/57), RAC3在脑胶质瘤组织中的表达率显著高于癌旁组织, 差异具有统计学意义(P<0.01)。转染siRNA后, 实验组和对照组U87MG细胞中RAC3 mRNA表达量分别为1.23±0.20和0.43±0.12, RAC3蛋白表达量分别为1.19±0.11和...     

脑胶质瘤细胞Ｐ—糖蛋白表达及临床意义

目的　探讨脑胶质瘤细胞P -糖蛋白 (P -gp)的表达及临床价值。 方法　采用s -p免疫组织化学法 ,对 6 3例手术后脑胶质瘤标本石蜡切片 ,测定P -gp表达。 结果　 6 3例脑胶质瘤P -gp阳性率 39.6 8% (2 5 /6 3) ;8例化疗后复发脑胶质瘤P -gp阳性率 87.5 0 % (7/8) ;两者间存在显著差异 (P <0 .0 2 5 )。 6 3例脑胶质瘤病理分级中 ,P -gp阳性率Ⅱ级 2 5 .0 0 % ,Ⅲ级 38.46 % ,Ⅳ级 5 2 .39% ,病理分级与P -gp表达呈正相关 (P <0 .0 0 5 )。结论　P -gp表达可为原发性和获得性的 ,化疗能诱导或增强肿瘤耐药性 ,P -gp表达随脑胶质瘤恶性程度增强 ,可作为临床化疗中的重要指导性指标。     

体外调节Th1/Th2平衡治疗脑胶质瘤

目的 观察外源性细胞因子白细胞介素( IL)-2、干扰素(IFN)-γ和IL-4单克隆抗体(McAb)、IL-10 McAb在体外能否促使Th2型细胞因子分泌优势向Th1型逆转,抑制胶质瘤细胞生长。方法 体外培养大鼠C6胶质瘤细胞,加入外源性细胞因子共同孵育,光学显微镜观察细胞形态学变化,噻唑蓝(MTT)比色法检测细胞增殖抑制率,逆转录-聚合酶链反应(RT-PCR)检测Th1/Th2类细胞因子表达变化。结果 实验组较对照组细胞体积变小,生长密度低,细胞数目少;实验组与对照组比较细胞增殖抑制率分别为(26.41 ±3.60)％,其A值与对照组比较差异有统计学意义(P＜0.05)。对照组细胞只表达Th2类细胞因子,Th1类细胞因子表达缺如,实验组IFN-γ表达增加,IL-4和IL-10表达减弱,但是未见IL-2表达。结论 外源性细胞因子能够抑制脑胶质瘤细胞的生长,促进Th2型细胞因子分泌优势向Th1型逆转。     

Survivin mRNA表达对肝癌细胞凋亡和增殖及MDR-1影响的实验研究

目的　研究新的凋亡抑制因子survivinmRNA在肝细胞癌 (hepatocellularcarcinoma ,HCC)中的表达 ,测定肝癌的细胞凋亡和增殖及多药耐药基因mRNA在HCC中的表达 ,探讨survivin表达对肝癌细胞凋亡和增殖的影响及其与HCC多药耐药之间的相互关系。方法　 31例HCC癌组织及癌旁组织标本取自 2 0 0 0年 8月至 2 0 0 1年 4月浙江大学医学院第二附属医院。用逆转录聚合酶链反应(RT PCR)、TUNEL法和免疫组织化学法分别测定HCC癌组织及癌旁组织中survivinmRNA的表达情况、细胞凋亡指数和增殖指数及MDR 1mRNA的表达 ,分析在HCC中survivin表达与细胞凋亡和增殖之间的相互关系 ,探讨survivin表达在HCC发生发展中的作用及其与MDR 1mRNA表达之间的关系。结果　 31例肝癌组织中survivinmRNA阳性例表达率为 71% (2 2 / 31) ,阴性表达率为 2 9% (9/ 31) ;而癌旁正常组织中均无表达。肝癌标本中的凋亡指数为 :0 4 %～ 15 % ,平均值为 :(4 2 4± 3 92 ) %。Sur vivinmRNA阳性表达病例平均AI值为 (4 6 4± 4 30 ) % ,阴性表达平均AI值为 (3 36± 2 98) %。两者比较P >0 0 5 ,两组间无显著差异。肝癌标本中增殖指数为 :2 %～ 80 % ,平均值为 (2 6 2 1± 14 9) %。SurvivinmRNA阳性表达病例平均Ki 6 7LI值为 (30 32± 15 4     

恶性脑肿瘤中CD44内含子9及CD44v6mRNA的表达水平及临床意义

目的 了解CD44内含子9、CD44v6mRNA在恶性脑肿瘤中的表达水平及临床意义。方法 应用RT－PCR技术检测28例恶性脑膜瘤,25例脑神经胶质瘤及20例脑瘤旁组织中CD44内含子9、CD44v6mRNA的表达并对mRNA含量进行半定量分析。结果 CD44v6mNRNA在恶性脑膜瘤和脑神经胶质瘤的阳性表达率分别为92．9％（26／28）和88．0％（22／25）,明显于高瘤旁组织的5％（1／20）,差异有非常显著性意义（P＜0．01）,前二者相对含量（mass）值明显高于瘤旁组织。恶性脑膜瘤和脑神经胶质瘤CD44内含子9mRNA表达水平明显高于瘤旁组织（P＜0．01）。结论 CD44内含子9、CD44v6mRNA内含子9mRNA表达水平明显高于瘤旁组织（P＜0．01）。结论 CD44内含子9、CD44v6mRNA对辅助诊断恶性脑肿瘤有一定的意义。     

Prolonged survival of mice with established intracerebral glioma receiving combined treatment with peroxisome proliferator-activated receptor-gamma thiazolidinedione agonists and interleukin-2-secreting syngeneic/allogeneic fibroblasts

OBJECT: In this study the authors explored the benefits of treating C57B1/6 mice with an established intracerebral glioma by combining immunotherapy with interleukin (IL)-2-secreting syngeneic/allogeneic fibroblasts administered into the tumor bed along with the chemotherapeutic agent pioglitazone, a thiazolidinedione (TZD). The TZDs are agonists of the peroxisome proliferator-activated receptor-gamma. They have been found to exert antiproliferative effects on several transformed cell lines. Data from prior studies by these authors have revealed the immunotherapeutic properties of the IL-2-secreting fibroblasts in treating intracerebral gliomas in mice. METHODS: The sensitivity of GL261 glioma cells and primary astrocytes to pioglitazone was determined in vitro by incubating the cells with increasing amounts of the drug. Viability was assessed by measuring lactate dehydrogenase release, and effects on metabolism were determined by measuring superoxide production and levels of superoxide dismutase. The GL261 cells were injected intracerebrally into C57B1/6 mice, followed by treatment with pioglitazone either orally or intracerebrally into the tumor bed. The effect of the combined therapy was determined by injecting C57B1/6 mice with an established intracerebral GL261 glioma with IL-2-secreting allogeneic fibroblasts and pioglitazone directly into the tumor bed through a unique cannula system. Pioglitazone was found to induce cell death in GL261 glioma cells grown in vitro while causing only modest damage to astrocytes. The application of pioglitazone also resulted in a significantly greater induction of cellular superoxide in glioma cells than in astrocytes, which can activate apoptotic pathways. Pioglitazone administered intracerebrally (p < 0.05) but not orally was found to prolong survival in mice harboring an intracerebral glioma. Synergistic effects of combination therapy on prolonging survival were found in mice receiving both pioglitazone and IL-2-secreting fibroblasts (p < 0.005, compared with untreated animals). Pioglitazone induces metabolic and oxidative stresses that are tolerated by astrocytes but not glioma cells, which could account for selective vulnerability and increased sensitivity to IL-2, suggesting potential for the use of this Food and Drug Administration-approved drug in the treatment of brain tumors. CONCLUSIONS: The data indicate the beneficial effects of combination therapy using pioglitazone and immunotherapy in mice harboring intracerebral glioma.     

Thromboxane synthase inhibitors induce apoptosis in migration-arrested glioma cells

OBJECTIVE: Because of the wide dissemination of malignant glioma cells by the time that malignant glioma is diagnosed, anti-invasive strategies that are designed to limit their further spread may be of little value unless mechanisms of the invasive cascade can be used to render invasive cells susceptible to cytoreductive treatments. We recently determined that elevated thromboxane synthase gene expression and enzymatic activity are associated with a highly migratory phenotype of glioma cells in vitro and that specific inhibitors of this enzyme block cell migration. Interference with this inherent phenotype of malignant gliomas also affects glioma cell proliferation and apoptosis. METHODS: To study the effect of thromboxane synthase inhibitors on motility, metabolic activity, and cell death, we used five human glioma cell lines, four glioblastoma-derived, low-passage cell cultures, normal human astrocytes, and fibroblasts. Motility was measured in a monolayer migration assay. Caspase activation as an early event in apoptotic cell death was assessed using a caspase 3 cleavage assay. Intracellular deoxyribonucleic acid (DNA) fragmentation was detected by enzyme-linked immunosorbent assay quantification of histone-complexed DNA. Subsequent cell death was scored by trypan blue exclusion. RESULTS: In this study, we demonstrate that the treatment of human glioma cells with the specific thromboxane synthase inhibitor furegrelate leads first to caspase activation (detectable 6 h after treatment), then to DNA fragmentation (24-48 h after treatment) and subsequent cell death. Caspase inhibitors abrogate this effect. Furthermore, the inhibition of thromboxane synthase by furegrelate increases cells' susceptibility to the induction of DNA fragmentation by camptothecin, etoposide, N,N'-bis(2-chloroethyl)-N-nitrosourea, and anti-CD95 antibodies. No induction of apoptosis was observed in normal astrocytes and fibroblasts. CONCLUSION: These data indicate that thromboxane synthase may represent a vortex of divergent signaling cascades that regulate motility and apoptosis in glioma cells. This paradigm may offer a novel perspective in the treatment of patients with malignant gliomas.     

Administration of interleukin-12 and -18 enhancing the antitumor immunity of genetically modified dendritic cells that had been pulsed with Semliki forest virus-mediated tumor complementary DNA

OBJECT: Immunogene therapy for malignant gliomas was further investigated in this study to improve its therapeutic efficacy. METHODS: Dendritic cells (DCs) were isolated from bone marrow and pulsed with phosphate-buffered saline or Semliki Forest virus (SFV)-mediated 203 glioma complementary (c)DNA with or without systemic administration of interleukin (IL)-12 and IL-18 to treat mice bearing the 203 glioma. To study the immune mechanisms involved in tumor regression, the authors investigated tumor growth of an implanted 203 glioma model in T cell subset-depleted mice and in interferon (IFN) gamma-neutralized mice. To examine the protective immunity produced by tumor inoculation, a repeated challenge of 203 glioma cells was given by injecting the cells into the left thighs of surviving mice and the growth of these cells was monitored. The authors demonstrated that the combined administration of SFV-cDNA, IL-12, and IL-18 produced significant antitumor effects against the growth of murine glioma cells in vivo and also can induce specific antitumor immunity. The synergic effects of the combination of SFV-cDNA, IL-12, and IL-18 in vivo were also observed to coincide with markedly augmented IFN-gamma production. The antitumor effects of this combined therapy are mediated by CD4+ and CD8+ T cells and by NK cells. These results indicate that the use of IL-18 and IL-12 in DC-based immunotherapy for malignant glioma is beneficial. CONCLUSIONS: Immunogene therapy combined with DC therapy, IL-12, and IL-18 may be an excellent candidate in the development of a new treatment protocol. The self-replicating SFV system may therefore provide a novel approach for the treatment of malignant gliomas.     

转染连接蛋白基因对胶质瘤细胞细胞间隙连接通讯及其生长变化的研究

目的　研究连接蛋白 (Cx)基因对C6胶质瘤细胞的增殖抑制及细胞间隙连接通讯(GJIC)的作用 ,探讨以Cx43基因治疗胶质瘤的可行性。方法　将含Cx43cDNA的质粒以Lipofec tamine介导转染Cx43表达缺失的C6胶质瘤细胞 ,通过Northern印迹杂交、原位杂交及免疫组织化学染色检测Cx43mRNA及蛋白表达 ,划痕标记荧光染料示踪技术 (SLDT)检测GJIC ,MTT法测定细胞增殖率 ,核仁组成区嗜银蛋白 (AgNOR)染色检测细胞增殖活性 ,TUNEL法检测细胞凋亡。结果　转染后C6细胞有不同程度的Cx43mRNA及蛋白表达和GJIC恢复。表达Cx43的克隆细胞增殖明显下降 ,培养 2～ 6d时 ,每天除C6组与空载组差异无显著性外 ,其余各组差异均有非常显著性 (P <0 .0 1) ,但细胞凋亡并未增加。结论　Cx43基因及GJIC在恶性胶质瘤的发生发展过程中起重要作用 ,可能成为恶性胶质瘤基因治疗的优选靶的之一。     

Increased invasive capacity of connexin43-overexpressing malignant glioma cells

OBJECT: Malignant glioma cells, similar to astrocytes, express connexin43 (Cx43) universally but at widely varied levels. Data from previous studies have demonstrated that malignant glioma cells form functional gap junction channels among themselves as well as with astrocytes and that such a communication has the potential to modulate the phenotypic characteristics of astrocytes. Recently, gap junctions have been demonstrated to play a role in the invasive phenotype of malignant gliomas. In this study, the authors have further investigated the motility and invasion ability of Cx43-overexpressing and Cx43-deficient malignant glioma cells. METHODS: Using a standard invasion system of a Matrigel transwell invasion chamber, the authors found that the number of Cx43-transfected C6 glioma cells (C6-Cx43 cells) migrating through the Matrigel-coated membrane was similar to that of mock-transfected control cells (C6-mock cells) during the first 24 hours, but increased significantly thereafter. When these cells were cocultured with astrocytes, the number of invading C6-Cx43 cells was more than threefold greater than the number of invading C6-mock cells. Results of an in vitro cell motility assay also demonstrated that C6-Cx43 cells were more motile and scatter-active than C6-mock cells. Furthermore, zymographic analysis of MMPs, an important determinant in glioma invasion, demonstrated that the amounts of MMP-2 and MMP-9 in culture medium collected from C6-Cx43 cells were orders of magnitude higher than those from C6-mock cells. In addition, BB-94, a synthetic MMP inhibitor, significantly inhibited C6-Cx43 cell invasion. CONCLUSIONS: The overexpression of gap junction proteins in glioma cells and the intercellular communication between tumor and nontumor glia cells may play important roles in the facilitation of glioma cell invasion.     

Oncogenes and growth factors in gliomas

The activation of cellular proto-oncogenes is related to the genesis and progression of neoplasias. Protein growth factors and their cellular receptors have been identified as products of some proto-oncogenes. The role of epidermal growth factor receptor (EGFr) in gliomas is presented. The expression of mRNA for platelet-derived growth factor (PDGF) and PDGF B-type receptor (PDGF-rec-B) in gliomas is analyzed. Gliomas express "in vivo" PDGF.B and PDGF-rec-B mRNAs. PDGF.B mRNA levels correlate with GFAP mRNA and does not correlate with the degree of malignancy. This is in agreement with the hypothesis of an autocrine growth stimulation in gliomas. However some findings seem to indicate that in these tumors the PDGF-rec-B is preferentially expressed by vascular elements. Thus, also a paracrine loop for endothelial cell growth stimulation may be suggested in malignant gliomas.