DEVELOPMENT OF A NEW IMMUNOTHERAPY FOR GLIOMA PATIENTS USING MOLECULER BIOTHCHNOLOGICAL TECHNIQUES

为了提高LAK治疗的效率，我们首先通过亲和素-生物素法研究LAK细胞与抗egf - r单克隆抗体结合的可能性。该方法可使胶质瘤患者淋巴细胞LAK活性提高约1.2倍。我们也可以通过这种方法从患者的辅助T细胞中产生足够的LAK细胞。由此结果提示LAK细胞与抗egf - r抗体联合使用可能会促进LAK对胶质瘤患者的治疗。其次，我们检测了与erbB2、TGF α、c-fos和c-myc的感觉mRNA互补的18bp寡脱氧核苷酸对胶质瘤细胞生长的影响。我们用聚合酶链反应研究了这些基因在培养的人胶质瘤细胞系中的表达。我们在7个胶质瘤细胞系中检测到4个TGF - α的mRNA转录物。与TGF α mRNA互补的反义寡核苷酸在体外被有效地整合到胶质瘤细胞中，动力学研究表明，与反义寡核苷酸孵育48小时内，最大的摄取发生。人类胶质瘤细胞系暴露于针对第一起始密码子的反义寡脱氧核苷酸中，以时间和剂量依赖的方式抑制细胞增殖。在人胶质瘤细胞系SNB-19中添加1微米TGF α特异性反义引物，可抑制胶质瘤生长80%。这种影响是饱和的和特定的。反义引物指向该mRNA的另一个位点，对抑制胶质瘤生长无效。反义引物和正引物均不能抑制胶质瘤细胞的生长。与此相反，相应的义寡聚物既不抑制tgf - α蛋白的合成，也不抑制胶质瘤细胞的生长。其他反义寡核苷酸对胶质瘤细胞无影响。综上所述，这些结果清楚地支持tgf - α蛋白在增殖过程中的作用，并表明诱导蛋白

To promote the efficiency of LAK therapy, at first, we investigated the possibility of the combination of LAK cells with the anti-EGF-R monoclonal antibody by the avidin-biotin method. By this method, we could enhance the LAK activity of lymphocytes from glioma patients about 1.2 times. We could also produce sufficient LAK cells from patients'helper T cell by theis procedure. From this result, it was suggested that LAK therapy for glioma patients might be promoted by the combined use of LAK cells with the anti-EGF-R antibody. Secondaly, we examined the effect of 18-bp oligodeoxynucleotides complementary to the sense mRNA of erbB2, TGF alpha, c-fos, and c-myc upon glioma cell growth. We investigated the expression of these genes within cultured human glioma cell lines by using ploymerase chain reaction. We could detct mRNA transcripts of TGF alpha in 4 out of 7 glioma cell lines. The antisense oligonucleotieds complementary to TGF alpha mRNA were efficiently incorporated into glioma cells in vitro and the kinetic study showed thast maximum uptake occurred within 48 hours of incubation with antisense oligomers. Exposure of human glioma cell lines to antisense oligodeoxylnucleotides targeted against first initiation codon inhibited cell proliferation in a time and dose dependent fashion. The addition of 1microM TGF alpha-specific antisense primer to the human glioma cell line SNB-19 resulted in an 80% inhibition in glioma growth. This effect was saturable and specific. Antisense primers directed to another sites of this mRNA were not effective in suppressing glioma growth Neither antisense or sense primers inhibited the growth of our glioma cells. In contrast to this, the corresponding sense oligomers inhibited neither syntheses of TGF-alpha protein nor glioma cell growth. Other antisense oligonucleotides could not affect glioma cells. Taken together, these results clearly support a role of TGF-alpha protein in the proliferation process and show that inducivle protein