| Project/Area Number |
09670186
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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 |
Human pathology
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| Research Institution | HIROSHIMA UNIVERSITY |
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Principal Investigator |
YOKOZAKI Hiroshi Hiroshima University Faculty of Medicine, Assistant Professor, 医学部, 講師 (10200891)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 1998: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1997: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | Human / Digestive system / Gastric cancer / Colorectal cancer / Cyclin E / Transfection / Antisense vector / Oncogene / アンチセンスペクター / 細胞培養 / 遺伝子導入 / 遺伝子発現 |
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| Research Abstract |
1. Construction of cyclin E expression vector and introduction to mouse NIH/3T3 fibroblast
(1) A 2.5 Kbp fragment of cyclin E cDNA that contained its total open reading frame was subcloned in to EcoRI site of mammalian expression vector pCDNA3 with cytomegalovirus promotor.
(2) The cyclin E expression vector or mock pCDN3 were transfected to mouse NTH/3T3 fibroblast by lipofection method. After G418 selection, each 10 clones were recovered. Integration of the vectors and expression of recombinant cyclin E protein were confirmed by Southern and Western blot analysis, respectively.
(3) Significant difference in growth rates were not observed between cyclin E transvectant and mock transfectant. No transformation focus was detected in cyclin E transfectants.
2. Construction of cyclin E antisense vector and introduction to human gastric cancer cell lines.
(1) A 11theta bp cyclin E cONA fragment was subcloned in antisense direction to pCDNA3 vector.
(2) antisense cyclin E vector was transfected into human gastric cancer cell lines (TMK.-1, MKN-7, MKN-28 and MKN-74). Each 10 G418 resistant clones were recovered.
(3) Significant difference in growth rates were not observed between antisense cyclin E transfectant and mock transfectant.
These in vitro observations in the present research project provided any direct evidence supporting the idea that cyclin E can act as oncogene in human gastrointestinal carcinogenesis.
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