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Inhibition of glioma angiogenesis and invasion by anti-microtubule agent in a rat brain tumor model.

在大鼠脑肿瘤模型中抗微管剂抑制神经胶质瘤血管生成和侵袭。

基本信息

批准号：
14571340
负责人：
YOSHIDA Daizo
金额：
$ 1.66万
依托单位：
Nippon Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14571340/
关键词：
brain tumor GFP tumor angiogenesis microtubule cell invasion glioblastoma glioblastoma グリオーマ血管新生 MMP SI-27 matrix metallo proteinase レーザー顕微鏡

项目摘要

We mainly achieved the following two investigations.(1)OBJECTIVE : We aimed to analyze the anti-invasive effect of the anti-matrix metalloproteinase (anti-MMP) agent SI-27 by quantitative tracking of enhanced green fluorescent protein(EGFP)-labeled human malignant glioma cell lines in a brain slice model. METHODS : Persistent expression of EGFP in human malignant glioma cell clones (U87MG, U251MG, and U373MG) was established with the use of the pEGFP-C1 vector. Tumor spheroid in 1 microl Matrigel was implanted into the caudate nucleus-putamen of a severe combined immunodeficient mouse brain slice. To allow the quantitative assessment of tumor cell invasion, the invasion area index was measured on Days 1,3,5,and 7 with a fluorescence stereomicroscope and an image analyzer in the presence of various concentrations of SI-27(0,1,10,50,or 100 microg/ml). RESULTS : In the control group (0 microg/ml), all glioma cell lines invaded in a fingerlike fashion and reached the contralateral hemisphe … More re through the corpus callosum. SI-27 at concentrations of 10,50,and 100 microg/ml significantly suppressed the invasion area index on Days 5 and 7 in a dose-dependent manner, whereas 1 microg/ml had no effect. Transmission electron microscopy and laser confocal microscopy indicated that the tumor cells had penetrated the brain slice and that the normal structural integrity of the brain was maintained until Day 7. CONCLUSION : This model enabled unequivocal periodic tracking of individual invading tumor cells in normal brain. The significant suppression of glioma cell invasion by noncytotoxic concentrations of SI-27 indicates that anti-MMP treatment may represent an important future therapeutic strategy for malignant cerebral neoplasms.(2)OBJECTIVE : The matrix metalloproteinase(MMP) inhibitor SI-27 has undergone extensive development because of its effectiveness against glioma invasion and angiogenesis. However, previous studies have been performed in vitro. The present work investigates the potential of SI-27 to inhibit tumor invasion, slow angiogenesis, and prolong survival in rodent brain tumor models. METHODS: Stable enhanced green fluorescent protein-expressing clones of a human malignant glioma cell line, U251MG, were stereotactically xenografted into the periphery of the anterior striatum and corpus callosum of Fischer 944 rats after immunosuppression with cyclosporin A, SI-27(1 or 10 mg/kg) or carrier solution was administered on three successive days by intraperitoneal injection, and tumor invasion and angiogenesis were assessed 3 weeks later by quantitative image analysis. This was performed on whole brain sections analyzed either by direct observation of enhanced green fluorescent protein-expressing glioma cells or by additional immunohistochemistry to detect the endothelial cells with anti-factor VIII monoclonal antibody In situ zymography on frozen sections was used to detect MMP activity. RESULTS : The group receiving a total of 30 mg/kg showed a statistically significant (P<0.001) increase in survival time compared with the controls receiving carrier (median survival, 47.3 versus 32.6 d). There was also a decrease in MMP activity, tumor cell invasion, and neovascularization. In contrast, animals given 3 mg/kg did not show these differences. CONCLUSION : Systemic administration of the anti-MMP agent SI-27 is effective in the treatment of glioma in an animal model. Less

我们主要完成了以下两项研究。(1)目的：我们旨在通过脑切片模型中增强型绿色荧光蛋白(EGFP)标记的人恶性胶质瘤细胞系的定量追踪来分析抗基质金属蛋白酶(抗MMP)剂SI-27的抗侵袭作用。方法：使用 pEGFP-C1 载体建立 EGFP 在人恶性胶质瘤细胞克隆（U87MG、U251MG 和 U373MG）中的持续表达。将1微升基质胶中的肿瘤球体植入严重联合免疫缺陷小鼠脑切片的尾状核壳核中。为了定量评估肿瘤细胞侵袭，在不同浓度的SI-27（0、1、10、50或100微克/毫升）存在下，使用荧光立体显微镜和图像分析仪在第1、3、5和7天测量侵袭面积指数。结果：在对照组（0微克/毫升）中，所有胶质瘤细胞系均以手指状方式侵入，并通过胼胝体到达对侧半球。浓度为10、50和100μg/ml的SI-27以剂量依赖性方式显着抑制第5天和第7天的侵袭面积指数，而1μg/ml则没有效果。透射电子显微镜和激光共聚焦显微镜表明，肿瘤细胞已穿透脑切片，并且大脑的正常结构完整性一直保持到第 7 天。结论：该模型能够对正常大脑中单个入侵的肿瘤细胞进行明确的周期性跟踪。非细胞毒性浓度的SI-27对胶质瘤细胞侵袭的显着抑制表明，抗MMP治疗可能是未来治疗恶性脑肿瘤的重要策略。(2)目的：基质金属蛋白酶(MMP)抑制剂SI-27因其有效对抗胶质瘤侵袭和血管生成而得到广泛的开发。然而，之前的研究都是在体外进行的。目前的工作研究了 SI-27 在啮齿动物脑肿瘤模型中抑制肿瘤侵袭、减缓血管生成和延长生存期的潜力。方法：将稳定表达增强型绿色荧光蛋白的人恶性胶质瘤细胞系U251MG克隆立体定向异种移植到Fischer 944大鼠的前纹状体和胼胝体周围，并用环孢素A、SI-27（1或10 mg/kg）或载体溶液进行免疫抑制，对3只大鼠进行免疫抑制。连续几天腹腔注射，3周后通过定量图像分析评估肿瘤侵袭和血管生成。这是在全脑切片上进行的，通过直接观察表达增强的绿色荧光蛋白的神经胶质瘤细胞或通过额外的免疫组织化学来检测具有抗因子VIII单克隆抗体的内皮细胞，使用冷冻切片上的原位酶谱法来检测MMP活性。结果：与接受载体的对照组相比，接受总共 30 mg/kg 剂量的组的生存时间显着延长（P<0.001）（中位生存期为 47.3 天对 32.6 天）。 MMP 活性、肿瘤细胞侵袭和新血管形成也有所下降。相比之下，给予 3 mg/kg 剂量的动物则没有表现出这些差异。结论：抗 MMP 药物 SI-27 的全身给药可有效治疗动物模型中的神经胶质瘤。较少的