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

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基因转染人肿瘤源性细胞。然而，这些转染的肿瘤细胞生长不好。这种生长抑制活性依赖于IL-12基因的半部分p35基因。因此，在以下实验中使用亲本肿瘤细胞。进行人肿瘤细胞的大量培养。用pH3.4的柠檬酸-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。通过HPLC系统提取低分子肽（M.W.1000 ± 6，500道尔顿）。将获得的多个峰中的一个峰添加到正常人细胞中。孵育几个小时后，提取m-RNA，以检查在8000个测试基因中有多少基因被上调或下调。大约200个基因的表达显著上调或下调。另一方面，上调的基因与肿瘤进展相关;下调的基因与针对抗肿瘤基因的阻遏基因相关。结果表明，该肽的特性与恶性肿瘤细胞的生长活性一致。下文的研究将集中在质谱仪测定的肽序列上。这也将是必要的分析这些巨大的基因表达的关系，以发展新的方法，多维度的统一。

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.