Analysis of the immunosuppressive state in patients with malignant brain tumors

恶性脑肿瘤患者免疫抑制状态分析

• 批准号: 11671362
• 负责人: NAGANUMA Hirofumi
• 金额: $ 2.3万
• 依托单位: Yamanashi Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671362/
• 关键词: malignant glioma; thrombospondin; transforming growth factor-β; synthetic peptide; cell motility; enzyme-linked immunosorbent assay; ELISA; 腫瘍浸潤能

Background : Malignant glioma cells secreted both thrombospondin-1 (TSP-1) and transforming growth factor-b (TGF-β) and the expression of both cytokines correlated with malignancy of gliomas. (Neuropathology 20 : 161, 2000) TSP-I has a potential to activate a latent form of TGF-β.Purpose : Experiments were performed to elucidate whether 1) TSP-1 secreted by malignant glioma cells participates in a latent TGF-β activaticn, and 2) a synthetic peptide which are found In TSP-1 moleoule and has a function of adhesion Can inhibit a motility of glioma cells. We established an ELISA for measurement of TSP-1 secretion by malignant glioma Cells.Results :1) Role of TSP-1 in latent TGF-β activation : Neutralizing anti-TSP-1 mouse monoclonal antibody or rabbit polyclonal antibody was added to culture medium of malignant glioma cells. The active form of TGF-β In the supernatants was measured by ELISA Addition of the antibodies resulted in a decrease of the active TGF-β in the supernatants in a dose-related manner. This Indicates that TSP-1 participates in the latent TGF-b activation. (Neurol Med Chir (Tokyo), in press)2) Inhibition of motility of glioma cells by a synthetic peptide : To examine the role of TSP-1 in motility of glioma cells, malignant glioma cells wae treated with a synthetic peptide which includes three adhesion sites in TSP-1 molecule. The motility of glioma cells was Inhibited by the peptide in a dose-related manner. The result agrees with the previous observation that transfection of antisense TSP-1 cDNA into glioma cells resulted in a decrease of motility of glioma cells. (Manuscript submitted)3) Measurement of TSP-1 in culture supernatants by ELISA : A sandwich ELISA using mouse anti-TSP-1 antibody and rabbit antibody was established Concentrations of TSP-1 in culture supernatants of 9 glioma cell lines were 43 - 2431 ng/1 x 10^6 cells/24 hours (643 ± 838 ; mean ± SD), while that of malignant melanoma (DFB) was 7.5 ng/1 x 10^6 cells/24 hours.

背景资料：恶性胶质瘤细胞同时分泌血小板反应蛋白-1（TSP-1）和转化生长因子-β（TGF-β），两者的表达与胶质瘤的恶性程度相关。（神经病理学20：161，2000）TSP-1具有激活TGF-β的潜在形式的潜力。目的：进行实验以阐明1）恶性胶质瘤细胞分泌的TSP-1是否参与潜在的TGF-β激活，以及2）在TSP-1分子中发现的具有粘附功能的合成肽是否能够抑制胶质瘤细胞的运动。结果：1）TSP-1在TGF-β潜在活化中的作用：在恶性胶质瘤细胞培养液中加入中和性抗TSP-1的鼠单克隆抗体或兔多克隆抗体。通过ELISA测量上清液中TGF-β的活性形式。抗体的添加导致上清液中活性TGF-β以剂量相关的方式减少。这表明TSP-1参与了潜在的TGF-β激活。（Neurol Med Chir（Tokyo），出版中）2）通过合成肽抑制神经胶质瘤细胞的运动性：为了检测TSP-1在神经胶质瘤细胞运动性中的作用，用合成肽处理恶性神经胶质瘤细胞，所述合成肽在TSP-1分子中包括三个粘附位点。该肽能抑制胶质瘤细胞的运动，并呈剂量相关性。这一结果与以前的观察结果一致，即反义TSP-1 cDNA转染胶质瘤细胞导致胶质瘤细胞运动能力下降。3）ELISA法测定培养上清中TSP-1的含量：建立了鼠抗TSP-1抗体和兔抗TSP-1抗体的双抗体夹心ELISA法。9种胶质瘤细胞系培养上清中TSP-1的含量为43 - 2431 ng/1 × 10^6细胞/24小时（643 ± 838 ;平均值±标准差），而恶性黑色素瘤（DFB）为7.5 ng/1 x 10^6个细胞/24小时。

项目成果

佐々木厚: "Participation of thrombospondin-1 in the activation of latent transforming growth factor-β in malignant glioma cells"Neurol Med Chir (Tokyo). (in press).

Atsushi Sasaki：“血小板反应蛋白-1 参与恶性神经胶质瘤细胞中潜在转化生长因子-β 的激活”Neurol Med Chir（东京）（出版中）。