|Budget Amount *help
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2005: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2004: ¥2,700,000 (Direct Cost: ¥2,700,000)
1)Production of EGF family growth factors, antimicrobial proteins, and polyclonal antibodies of them
We produced recombinant EGF family growth factors such as TGF - α, amphiregulin, epiregulin, HB-EGF, and human beta-defensin 1,2,3,4 and CAP18 which were antimicrobial peptides. We also made polyclonal antibodies using these recombinant proteins.
2)Analysis of innate immunity mechanism of keratinocyte
(1)We analyzed innate immunity mechanism of keratinocyte using recombinant proteins. We added recombinant EGF family growth factors into culture medium of keratinocytes, and mRNA expression of antimicrobial peptides was analyzed by RT-PCR and real-time PCR.
(2)We also investigated which kind of gene was induced by bacterial antigens such as staphylococcus aureus and a hemolytic streptococcus using real-time PCR.
(3)We stimulated keratinocytes with peptideglycan, poly(I : C), LPS, imidazoquinoline, CpG DNA and examined what kind of cytokines, chemokines and antimicrobial peptides were induced.
3)Expressions of antimicrobial proteins in mouse wound model
(1)We made wounds (8mm in diameter) on backs of mice and excised them 6, 12, 24hrs, 2, 3, 4, 7days respectively. Half of each specimen were fixed with 10% formalin and the others were frozen. Folmalin sections (4-6μm in thickness) were stained with H-E and frozen sections were stained immunohistochemically with anti-mouse beta-defensin 1, 2, 4, 6 antigens and anti-CRAMP antigen.
(2)Mouse beta-defensin 2 was found almost all parts of skin around the wound, but other antimicrobial peptides were found in limited parts of wound margine such as epidermis, dermis, and hair follicles. These peptides expressed from just after injury to 2nd or 4th day.