Analysis of MHC-restricted tumor rejection antigen induced by IL-12.

IL-12 诱导的 MHC 限制性肿瘤排斥抗原分析。

• 批准号: 11671973
• 负责人: UMEZU Yasuiki
• 金额: $ 2.56万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671973/
• 关键词: Human Tumor Cells; MHC class I; IL-12; Tumor Specific Antigen; Antigen Peptides; Gene Profile; CTL; Tumor Specific Antiqen; Cytokine(S); PBMC(3); Tumor Speafic Autigen

Genetic effects of human-tumor cells derived peptides were investigated. Because IL-12 gene transfected tumor cells highly expressed MHC class I molecules, IL-12 gene was transfected into human tumor derived cells. However, those transfected tumor cells did not grow well. This growth inhibitory activity depends on p35 gene which was a half part of IL-12 gene. So, parental tumor cells were used in the following experiments. Mass-culture of human tumor cells was done. After cell surface peptides were collected by pH3.4 citrate-phosphate buffer solution, crude samples were filtered by specific membrane systems. Low molecular peptides (M.W.1000〜6,500 Dalton) were extracted by HPLC system. One peak among multiple peaks obtained was added to normal human cells. After incubation for several hours, m-RNAs were extracted to examine what and how many genes were up- or down-regulated in eight thousand gene tested. The expression of about two hundred genes was significantly up- or down-regulated. The up-regulated genes were associated with tumor progression, in the other hand ; the down-regulated genes were associated with repression genes against anti-tumorigenic genes. As a result, the character of this peptide was consistent with the growth activity of malignant tumor cells. The investigation hereafter will be focused on the peptides sequences by mass spectrometer. It will be also necessary for analysis of the relations of these huge gene expressions to develop the new methods for multi-dimensional unification.

研究了人肿瘤细胞衍生肽的遗传效应。由于IL-12基因转染的肿瘤细胞高度表达MHC I类分子，因此将IL-12基因转染到人肿瘤源性细胞中。然而，转染后的肿瘤细胞生长不佳。这种生长抑制作用依赖于p35基因，它是IL-12基因的一半。因此，本实验采用亲代肿瘤细胞。进行了人肿瘤细胞的大规模培养。用pH3.4柠檬酸盐-磷酸盐缓冲液收集细胞表面肽后，用特定的膜系统过滤粗样品。用高效液相色谱法提取低分子多肽（分子量1000 ~ 6500道尔顿）。将获得的多个峰中的一个峰添加到正常的人细胞中。在孵育数小时后，提取m-RNAs，以检查在8000个被测试的基因中哪些基因被上调或下调，以及有多少基因被上调或下调。大约200个基因的表达明显上调或下调。另一方面，上调基因与肿瘤进展有关；下调基因与抗肿瘤基因的抑制基因相关。结果表明，该肽的特性与恶性肿瘤细胞的生长活性一致。今后的研究将集中在多肽序列的质谱仪。分析这些巨量基因表达之间的相互关系，建立多维统一的新方法也是必要的。