Diagnostic and therapeutic studies of ganglioside <GD_2> in human neuroectodermal tumors

本课题旨在研制抗神经节苷<GD_2>的单克隆抗体，为脑肿瘤的诊断和治疗开辟新的途径。神经节苷脂<GD_2>是胎儿脑和神经外胚层肿瘤表达的癌胎抗原。牛脑神经节苷<GD_(1b)>经牛肝< β > -半乳糖苷酶转化为<GD_2>。<GD_2>采用Sephadex G-25柱层析纯化。纯化的<GD_2>在完全弗氏佐剂和鸡蛋白蛋白中乳化。每周经皮和腹腔注射该抗原，持续一个月。免疫小鼠血清中未检出与<GD_2>反应的抗体。免疫小鼠脾细胞与小鼠骨髓瘤细胞（NS-1）融合。用低氧-蒽醌-氨基蝶呤-胸苷（HAT）培养基筛选杂交细胞。菌落形成细胞很少，抗体产生细胞无法获得。另一方面，用KNS-42人胶质瘤细胞系免疫小鼠的NS-1与脾脏细胞的杂交细胞获得了许多杂交瘤细胞系。其中一种杂交瘤细胞系产生对细胞表面蛋白（分子量：65000和66000）反应的抗体。体外培养的胶质瘤细胞的表面蛋白定位于丝状突起、皱褶膜和粘附斑块。正常人脑和胶质瘤的免疫组化研究表明，该抗体对星形胶质细胞和良性星形细胞瘤细胞有特异性反应。神经元和其他间充质细胞完全未染色。目前正在研究表面蛋白的功能作用。溴脱氧尿苷（BrdU）摄取研究显示，BrdU摄取高的病例预后较差。甘露醇对血脑屏障（BBB）破坏的研究表明，正常脑屏障比肿瘤脑屏障破坏更严重。颈动脉内灌注甘露醇打开血脑屏障可能对正常脑有危害。我们应该继续与脑肿瘤的斗争，建立有效的控制肿瘤的方法。少

The research project is to produce monoclonal antibody against ganglioside <GD_2> and to develop new diagnostic and therapeutic methods for brain tumors. Ganglioside <GD_2> is an oncofetal antigen expressed by fetal brain and neuroectodermal tumors. Bovine brain ganglioside <GD_(1b)> was extracted and converted to <GD_2> with bovine liver <beta> -galactosidase. <GD_2> was purified with Sephadex G-25 column chromatography. The purified <GD_2> was emulsified in complete Freund's adjuvant and chicken egg albumin. The antigen was weekly injected to mice intracutaneousely and intraperitoneally for a month. Any antibody reacting to <GD_2> was not detected in the sera of immunized mice. The spleen cells of immunized mice were fused to mouse myeloma cells(NS-1). The hybrid cells were selected with hypox-anthine-aminopterin-thymidine(HAT) media. Colony forming cells were very few and antibody producing cells were unable to obtain. On the other hand, many hybridoma cell lines were obtained from … More the hybrid cells between NS-1 and spleen cells of mice which were immunized with KNS-42 human glioma cell line. One of the hybridoma cell lines produces antibody reacting to cell surface proteins ( molecular weight: 65000 and 66000). The surface proteins localised in filopodea, ruffling membrane and adhesion plaque of cultured human glioma cells. Immunohistochemical studies on normal human brain and glioma revealed that the antibody specifically reacted to astrocytes and benign astrocytoma cells. Neurons and other mesenchymal cells were absolutely unstained. The functional roles of surface proteins are now studying.The proliferative activities analysed with bromodeoxyuridine(BrdU) uptake study revealed that the cases with high uptake of BrdU were poor outcome.The studies on blood brain barrier(BBB) disruption with mannitol revealed that the barriers of normal brain were more heavily destructed than those of tumors. BBB opening with intracarotid mannitol infusion may be hazardous on normal brain of patient.We should to keep up the fight against brain tumors and establish the effective method of controlling tumors. Less