Genetic diagnosis of gliomas and its clinical application

我们已经针对胶质瘤和髓母细胞瘤共有的抗原制备了小鼠单抗(ONS-M21)(BR J癌症68：831-837,1993)。然后，我们与中外制药有限公司合作，成功地实现了他们的抗体(Mol免疫)和他们的单链抗体的人源化。利用这些人源化抗体及其单链抗体，我们可能对肿瘤部位进行正确的诊断，并尝试将其应用于恶性胶质瘤患者的免疫治疗。目前已进入最后一步，即提取这些鼠源性单抗所识别的恶性胶质瘤相关抗原。在提取它们的抗原后，我们将确定它们的基因排列和表达机制。我们发表了两篇关于胶质细胞特异性启动子在胶质瘤细胞中选择性表达外源基因的论文(Jpn J Cancer Res 83：1244-1247,1992；J Neurosci Res 38：415-423,1994)。目前，我们在体内重复t…基因。更多的是在小鼠胶质瘤模型的这些体外数据下用小鼠髓鞘碱性蛋白(MBP)基因启动子直接表达毒性基因(J Neurosci Res 36：472-479,1993)。另一方面，为了了解胶质瘤细胞脑侵袭的细胞机制，我们用明胶亲和层析、肝素亲和层析、DEAE层析、羟基磷灰石凝胶渗透层析和磺丙基高效液相层析，从高侵袭性人胶质瘤细胞系T98G的无血清条件培养液中纯化了两种胶质瘤源性运动因子(GMF)分子，GMF-I和GMF-II。GMF-I和GMF-II均以浓度依赖的方式刺激T98G细胞迁移，GMF-I的活性约为GMF-II的5倍，C6胶质瘤细胞在重组基底膜Matrigel体外侵袭实验中显示出较高的侵袭力，向GMF迁移的强度较大，而A172和9L胶质瘤细胞和正常胶质细胞对Matrigel屏障的渗透较弱，向GMFS迁移的强度较小。这些结果表明，胶质瘤细胞对GMFS的迁移反应与侵袭性密切相关，提示GMFS在胶质瘤细胞侵袭过程中发挥着重要作用(Biochem BiPhys Commun 193：518-525,1993)。较少

We had already made mouse monoclonal antibodies(ONS-M21) for the antigens which were shared in both glioma and medulloblastoma(Br J Cancer 68 : 831-837,1993). Then we succeed in humanization of their antibodies (Mol Immunol, in press) and their single-chained antibodies in cooperation with Chugai Pharmaceutical Co.Ltd. Using these humanized antibodies and their single-chained antibodies, we might develop the correct diagnose of tumor sites, and will try to apply to immunotherapy of malignant glioma patients. And now it is in the last steps to extract the malignant glioma-associated antigens which were recognized with these mouse monoclonal antibodies. After extraction of their antigens, we will identify their gene arrangement and mechanisms of their expression.We publishied two papers about selective expression of foreign genes in glioma cells by glial-specific promoters (Jpn J Cancer Res 83 : 1244-1247,1992 ; J Neurosci Res 38 : 415-423,1994). At present, we repeats the in vivo gene t … More herapy in mouse glioma models under these in vitro data with the mouse myelin basic protein (MBP) gene promoters to direct toxic gene expression (J Neurosci Res 36 : 472-479,1993). Then, if we could elucidate the glioma-associated antigens accordig to our plans, we will analyze the promoters controlling their manifestation and will apply them in gene therapy with tissuespecificity in the future.On the other hand, to understand the cellular mechanism of brain invasion by glioma cells, two molecular species of glioma-derived motility factor (GMF), GMF-I and GMF-II,have been purified to homogeneity from the serum-free conditioned medium of a highly invasive human glioma cell line, T98G,by gelatin affinity chromatography and heparin affinity-, DEAE-, hydroxyapatite-, gel permeation- and sulfopropyl high performance liquid chromatography. GMF-I and GMF-II both stimulated the migration of T98G cells in a concentration-dependent manner, and the activity of GMF-I was about 5 times as strong as that of GMF-II.C_6 glioma cells, both of which showed high invasiveness in an in vitro invasion assay with reconstituted basement membrane, Matrigel, migrated to the GMFs with great intensity, while A172 and 9L glioma cells and normal glial cells, all of which weakly infiltrated the Matrigel barrier, migrated to the GMFs with much less intensity. These results indicate that migratory response of glioma cells to the GMFs correlates well with invasiveness, suggesting an important roles of the GMFs in the process of glioma cell invasion (Biochem Biophys Res Commun 193 : 518-525,1993). Less