| Project/Area Number |
11671700
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Otorhinolaryngology
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| Research Institution | DOKKYO UNIVERSITY SCHOOL OF MEDICINE |
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Principal Investigator |
ASAI Tadao Dokkyo University of Medicine, Oto rhino laryngology, Lecturer, 医学部, 講師 (90231860)
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| Co-Investigator(Kenkyū-buntansha) |
KANAYA Hiroaki Dokkyo University of Medicine, Oto rhino laryngology, Instructor, 医学部, 助手 (40265301)
HIRABAYASHI Hideki Dokkyo University of Medicine, Oto rhino laryngology, associate Professor, 医学部, 助教授 (00146185)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2000: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Keywords | head and neck malignant tumor / MHC class I / tumor associated antigen / ELISPOT Assay / antigen epitope / 頭頚部扁平上皮癌 / CTL / HLA A2 拘束性 / 樹状細胞 / acid stripping / Limitig dilution assay |
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| Research Abstract |
We induced CTL lines from peripheral blood mononuclear cells (PBMC) of healthy donors by using human head and neck malignant tumors. CTL lines were induced from PBMC with antigen presented cells pulsed with tumor associated antigens obtained from malignant cells. Evaluation of CTL lines were performed by limiting dilution assay (LDA) and ELISPOT assay. In ELISPOT assay, secretion of IFN-γ from antigen specific CTL lines was measured. After recognized that the CTL lines were specific for tumor cell lines, tumor associated antigens were separated into some fragments by High performance liquid chromatography (HPLC).
Frequencies of CTL lines were detected by LDA and ELISPOT assay at the same time. The results of both methods were almost equal. A lot of time and efforts were needed in LDA different from ELISPOT assay. ELISPOT assays were recognized as good method for evaluation of CTL lines induced from PBMC.
A number of fragments were obtained from tumor associated antigens by HPLC and presented on T2 cells. The CTL specific fragments were measured by ELISPOT assay. Analysis of CTL specific fragments was performed by mass spectrometry. Two proteins were detected, one consisted of ten amino acids, Met-Gly-Ala-Ala-Pro-Ser-(Leu or Ile)-(Leu or Ile)-Gly-(Leu or Ile). The other consisted of nine amino acids, Met-Gin-Ala-Pro-Ser-(Leu or Ile)-(Leu or Ile)-Gly-(Leu or Ile). These proteins were recognized new tumor specific antigens.
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