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

利用分子生物技术开发神经胶质瘤患者的新免疫疗法

• 批准号: 04454356
• 负责人: TAMURA Tetsuro
• 金额: $ 2.05万
• 依托单位: NIIGATA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04454356/
• 关键词: LAK CELLS; EGF-R; GLIOMA CELLS; ANTISENSE CLIGOMER; GROWTH FACTOR; TGF ALPHA; アンチセンス療法; オートクライン機構; TGF alpha; オリゴマー; neu; erbB2; 悪性グリオーマ; T細胞レセプター; LAK療法; サイトカイン産生能; ICAM-1

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

为了提高LAK细胞的治疗效率，我们首先采用亲和素-生物素法研究了LAK细胞与抗EGF-R单克隆抗体结合的可能性。用此方法可使胶质瘤患者淋巴细胞的LAK活性提高约1.2倍。我们也可以从病人的辅助性T细胞中产生足够的LAK细胞。提示LAK细胞与抗EGF-R抗体联合应用可能促进LAK治疗脑胶质瘤。其次，我们检测了与erbB 2、TGF α、c-fos和c-myc正义mRNA互补的18-bp寡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脑胶质瘤细胞系中的表达。我们可以在7个胶质瘤细胞系中的4个中检测到TGF α的mRNA转录。与TGF α mRNA互补的反义寡聚体在体外被有效地掺入胶质瘤细胞，动力学研究表明，与反义寡聚体孵育48小时内出现最大摄取。以第一起始密码子为靶点的反义寡核苷酸对人脑胶质瘤细胞株的增殖具有时间和剂量依赖性抑制作用。在人脑胶质瘤细胞系SNB-19中加入1 μ M TGF α特异性反义引物，导致胶质瘤生长抑制80%。这种效应是饱和的和特异的。针对该mRNA的另一个位点的反义引物在抑制胶质瘤生长中无效。反义或正义引物都不能抑制我们的胶质瘤细胞的生长。与此相反，相应的正义寡聚体既不抑制TGF-α蛋白的合成，也不抑制神经胶质瘤细胞的生长。其他反义寡核苷酸不能影响胶质瘤细胞。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

项目成果

Torp SH, Helseth E, Ryan L et al.: "Amplification of the epidermal growth factor receptor gene in Human gliomas." Anticancer Res. 11(6). 2095-2098 (1991)

Torp SH、Helseth E、Ryan L 等人：“人类神经胶质瘤中表皮生长因子受体基因的扩增。”

Alama A, Barbieri F, Bottini F et al.: "The use of antisense oligodeoxynucleotides (aODNs) for the therapy of cancer." Drugs Exp Clin Res. 17(12). 575-579 (1991)

Alama A、Barbieri F、Bottini F 等人：“使用反义寡脱氧核苷酸 (aODN) 治疗癌症。”

山中龍也 他: "悪性グリオーマのサイトカインネットワークの解析" 神経免疫研究. 5. 111-114 (1992)

Tatsuya Yamanaka 等人：“恶性神经胶质瘤中细胞因子网络的分析”《神经免疫学研究》5. 111-114 (1992)。

吉田 誠一: "脳腫瘍の免疫と分子生物学" 金芳堂, 8 (1992)

吉田精一：“脑肿瘤的免疫学和分子生物学”Kinhodo，8（1992）

Saxna A, Ali I U: "Increased expression of gnes from growth factor signaling pathways in glioblastoma cell lines" Oncogene. 7(2). 243-247 (1992)

Saxna A，Ali I U：“胶质母细胞瘤细胞系中生长因子信号通路中 gnes 的表达增加”Oncogene。