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

本研究的目的是制备抗神经节苷脂Gd_2和Gt的单抗，为脑肿瘤的诊断和治疗提供新的方法。神经节苷脂&lt；GD_2&gt；是一种由胎脑和神经外胚层肿瘤表达的致癌胎儿抗原。用牛肝β-半乳糖苷酶将牛脑神经节苷脂转化为-lt；GD_2&gt；。&lt；GD_2&gt；经Sephadex G-25柱层析纯化。纯化的&lt；GD_2&gt；在完全弗氏佐剂和蛋清蛋白中乳化。每周给小鼠皮下和腹膜内注射该抗原，持续1个月。免疫小鼠血清中未检测到与~lt；GD_2&gt；反应的抗体。将免疫小鼠的脾细胞与小鼠骨髓瘤细胞(NS-1)融合。用次氧-嘌呤-氨基蝶呤-胸腺嘧啶核苷(HAT)培养液筛选杂交瘤细胞。集落形成细胞很少，无法获得抗体产生细胞。另一方面，从…获得了许多杂交瘤细胞系用KNS-42人脑胶质瘤细胞株免疫的小鼠脾细胞与NS-1杂交瘤细胞。其中一个杂交瘤细胞系产生与细胞表面蛋白(分子量：65000和66000)发生反应的抗体。表面蛋白定位于培养的人脑胶质瘤细胞的丝足、皱褶膜和粘附斑中。对正常人脑和胶质瘤的免疫组织化学研究表明，该抗体与星形胶质细胞和良性星形细胞瘤细胞发生特异性反应。神经元和其他间充质细胞完全不染色。表面蛋白的功能作用正在研究中，用溴脱氧尿嘧啶核苷(BrdU)摄取分析其增殖活性，结果显示高摄取BrdU的患者预后较差；甘露醇破坏血脑屏障(BBB)的研究表明，正常脑屏障的破坏比肿瘤脑屏障的破坏更严重。颈动脉内输注甘露醇开放血脑屏障对患者正常脑组织有危险，应继续与脑肿瘤作斗争，建立有效的控制肿瘤的方法。较少

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