| Project/Area Number |
61480134
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Human pathology
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| Research Institution | Tokai University School of Medicine |
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Principal Investigator |
MORIUCHI Tetsuya Tokai University School of Medicine, Assistant Professor, 医学部, 講師 (20174394)
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| Co-Investigator(Kenkyū-buntansha) |
NAKANE Kazuo Tokai University School of Medicine, Professor, 医学部, 教授 (60164240)
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| Project Period (FY) |
1986 – 1987
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| Project Status |
Completed (Fiscal Year 1987)
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| Budget Amount *help |
¥6,200,000 (Direct Cost: ¥6,200,000)
Fiscal Year 1987: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1986: ¥4,700,000 (Direct Cost: ¥4,700,000)
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| Keywords | In situ hybridization / Nonradioactive probe / Hybridizaion / Oncogene / Cancer / Pathological diagnosis / Immunohistochemistry / Enzyme / DNAブローブ |
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| Research Abstract |
We have applied enzyme-immuno-histo-in situ hybridization (EIHISH) technique for the diagnosis and classification of leukemias and cancers. DNA fragments were chemically labeled with dinitrophenyl(DNP) residues. Or DNA fragments were UV- irradiared and adjacent thymidines on the nucleotide sequence were dimerized(T-T dimerization). These fragments were used as probes for non-radioactive in situ hybridization. We searched more better fixation of specimens, concentration of proteinase K for the exposure of target nucleic acids in the tissue, hybridization temperature, washing condition and probe size. These conditions were determined in the course of in situ hybridizations using various tissues and cells. Tissues and cells used in this experiments are as follows: human gastric cancers transplanted in nude mice, human T-cell leukemia cells, human livers infected with hepatitis B virus and Vero cells infected with herpes simplex virus type I. DNA probes used were c-myc, T-cell receptor beta chain, HTLV-1, hepatitis B virus, HSV-1 and alpha-fetoprotein.
Frozen-sectioned, cytocentrifuged or stamped specimens gave stronger positive signals than paraffin sections. Morphological architecture was best conserved in paraffin sections but poor in frozen sections. In frozen sections nonspecific staining was often observed especially in stromal tissues. These results indicate that EIHISH is applicable to cell biological experiments but immature tool for pathological diagnosis at present. Improvement of the sensitivity and the specificity in EIHISH will provide useful tool to pathological diagnosis based on oncogene probes.
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