|Budget Amount *help
¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2002: ¥1,000,000 (Direct Cost: ¥1,000,000)
The cosmid cassettes pAxCAw and pAxCALNLw, nuclear localizing signal-tagged Cre recombinase-expressing Ad (AxCANCre), control Ad Axlw, and the parent virus Ad5-d1X were all kind gifts from Dr. Izumu Saito (Tokyo University, Japan). The full-length coding regions of p53 cDNA was sub-cloned into the Ad cosmid cassette pAxCAw. Ad containing the CA promoter and p53 (AxCAp53) were generated using the COS-TPC method. The cosmid DNA was mixed with the EcoT22I-digested DNA-terminal Ad5-d1X protein complex, and used to co-transfect 293 cells. Recombinant viruses were generated through homologous recombination in 293 cells. Virus stocks were prepared using a standard procedure. Concentrated, purified virus stocks were prepared by CsC1 gradient, and the virus titer was checked by plaque formation assay.
We made small wounds exposing bare cartilage on the inner surface of rabbits' ears. We found reepithelization of the wound 3 weeks after operation, formation of keloid like lesions 4weeks after operation, and disappearance of the lesions 7weeks after operation. Histological findings of the lesions showed fibroblast proliferation, new vascular vessels generation, and lack of collagen deposition. The findings seemed to be characteristics of granulation tissue rather than keloid tissue.
The three keloid like lesions of each rabbit were injected p53 adenovirus-vector concentrated 1×10^6, 1X10^8, and 1X10^<10>PFU/ml respectively and compared with the other control lesion. But all lesions have necrosed within 1 week after adenovirus-vector injection and we couldn't continue the observation. We thought that he skin of the inner surface of rabbit ear is very thin and the necrosis of the lesions after injection occurred due to the adenovirus-vector's effect The concentration and quantity of the adenovirus-vector must be considered again.