| Budget Amount *help |
¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2002: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 2001: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Research Abstract |
TGF β RIwt gene was inserted in pAxCAwt vector, and it was amplified in E.coli using in vitro packaging kit GIGApacIII , and the cosmid DNA was co-trasfected with DNA-TPC into the HEK293 cells. The first virus soluton were obtaind 18/96 samples. In addition, the secondary virus solution were obtaind 5/18 samples by first virus infection to HEC293 cells. These 5 virus solution were confirmed that they did not destory HeLa cell, simultaneously. Also these virus solution were confirmed that the interminglement of the wild type virus was denied by using restriction enzyme analysis, and that the insert was rightly came with PCR analysis. Finary, the third virus solutions were obtaind also abouve methods. Human ovarian cancer cell lines without expressing TGF β RI were used the infection and growth suppression analysis. Cell proliferation was observed at 4 groups, A ; pAxCAwt-TGF β RI (+) , TGF β (+), B ; pAxCAwt-TGF β RI (+) , TGF β (-), C ; pAxCAwt-TGF β RI(-), TGF β (+), and D ; null dish. The clear difference between group A and D was observed in cell numbers at 12 days. On the other hands, somewhat cell proliferation suppression effect was observed in Group B and C. These were explaind by the suppression effect of TGF β function thourgh the way without TGF β RI, and non-specific protein synthesis suppresion mechanism in the condition of massive gene expression by pAxCAwt-TGF β RI vectors, respectively. Though virus solution with high titer had to be prepared more and more for the experiment of in vivo studys, our research suspected the possiblity to make a gene therapy model of ovarian cancer with mutation-replacement therapy by the TGF β RI gene introduction using adenovirus.
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