| Project/Area Number |
04670741
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
General surgery
|
|---|
| Research Institution | KYUSHU UNIVERSITY |
|---|
Principal Investigator |
INOUE Hiroshi MEDICAL INSTITUTE OF BIOREGULATION, LECTURE, 生体防御医学研究所, 助手 (90203249)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
NAKASHIMA Hideaki MEDICAL INSTITUTE OF BIOREGULATION, LECTURE, 生体防御医学研究所, 助手 (20253528)
ADACHI Masashi MEDICAL INSTITUTE OF BIOREGULATION, LECTURE, 生体防御医学研究所, 助手 (00253526)
UEO Hiroaki MEDICAL INSTITUTE OF BIOREGULATION, ASSOCIATE PROFESSOR, 生体防御医学研究所, 助教授 (70150430)
渋田 健二 九州大学, 生体防御医学研究所, 助手 (70253531)
|
|---|
| Project Period (FY) |
1992 – 1993
|
| Project Status |
Completed (Fiscal Year 1993)
|
| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1993: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1992: ¥1,000,000 (Direct Cost: ¥1,000,000)
|
|---|
| Keywords | Gene transfer / Gene therapy / Retroviral vector / Interleukin 2 / Mammalian expression vector / 置Gene transfer / レトロウィルスベクター / サイトカイン |
|---|
| Research Abstract |
Gene transfer method has now become a choice of treatment for various diseases, including cancer. To establish a human cell line which secretes sytokine, we isolated the interleukin 2(IL 2) cDNA, inserted it into a mammalian expression vector (pRc CMV) and transfected the resultant hybrid vector into a human gastric carcinoma cell line, MKN-28 with the Chen-Okayama method, and finally obtained 21 clones. The integration of the IL 2 cDNA in the hytbrid cells was confirmed with PCR method using primers (SP6 and T7 compatible) which flank the IL 2 cDNA.All 21 clones were shown to be integrated with a new IL2 cDNA.Immuno-histochemical analysis using anti IL 2 monoclonal antibody (Zymed laboratory) revealed a strong IL 2 expression widespread in the cytoplasm of the transfectant cells. Immunoblotting analysis for the detection of free IL 2 iN the aliquots of culture supernatent of the transfectants showed that more than 1 U/ml of IL 2 were secreted from the transfectants for 24 hrs. The IL 2 productin and secretion of the transfectant continued for 30-40 day, however, stopped after that.
We concluded that the gene transfer with plasmid vector was consistent and reliable method, however, its application for clinical treatment was quite difficult because its functional property was unstable. We, therefore, adopted the retroviral vector based-gene transfer method and promote recombination experiments.
|
