Analysis of the immunosuppressive state in patients with malignant brain tumors

背景：恶性胶质瘤细胞分泌凝血酶敏感蛋白-1(TSP-1)和转化生长因子-β(β-β)，二者的表达与胶质瘤的恶性程度有关。目的：探讨恶性胶质瘤细胞分泌的TSP-1是否参与潜在的转化生长因子-β的激活，以及TSP-1分子中一种具有黏附功能的合成肽是否能抑制胶质瘤细胞的运动。结果：1)TSP-1在潜在的转化生长因子-β激活中的作用：在恶性胶质瘤细胞的培养液中加入中和抗TSP-1鼠单抗或兔多克隆抗体。用酶联免疫吸附试验检测上清液中转化生长因子-β的活性形式。抗体的加入使上清液中转化生长因子-β活性降低，且呈剂量依赖关系。这表明TSP-1参与了潜在的转化生长因子-β的激活。(Neurol Med Chir(东京)出版)2)一种合成肽对胶质瘤细胞运动的抑制作用：为了研究TSP-1在胶质瘤细胞运动中的作用，用一种在TSP-1分子上含有三个黏附位点的合成肽处理恶性胶质瘤细胞。该多肽对胶质瘤细胞的运动有抑制作用，且呈剂量依赖性。这一结果与先前观察到的反义TSP-1基因导入脑胶质瘤细胞后导致细胞运动能力下降的结果一致。3)用小鼠抗TSP-1抗体和兔抗TSP-1抗体建立了双抗体夹心ELISA法测定培养上清液中TSP-1的浓度，9株胶质瘤细胞株培养上清液中TSP-1的浓度为43~2431 ng/1×10~(-6)个/24小时(643±838；均值±SD)，恶性黑色素瘤细胞系(DFB)培养上清液中TSP-1的浓度为7.5 ng/1×10~(-6)细胞/24小时。

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.