|Budget Amount *help
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1992 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1991 : ¥1,100,000 (Direct Cost : ¥1,100,000)
During the first year we attempted to obtain c-DNAs of types I,III,IV and V collagen, laminin and lysyl oxidase. Then these c-DNAs were reproduced in quantities. Among these c-DNAs, we used that of type IV collagen at first to investigate in vitro production of type IV collagen at the level of mRNA. Normal human epidermal keratinocytes (NHEK) were cultured in a MCDB culture medium adding the extract of human pituitary. At two conditions of confluent and subconfluent, RNA was extracted and Northern blotting was performed. While, at subconfluent, 2.1 kb sized band was apparent, at confluent, same band could not be found.
This result could be interpreted as follows ; 1. NHEK shows mRNA of type IV collagen and produces this type of collagen. 2. At the confluent condition, some cellular interaction or other causes inhibit the appearance of this gene. Secondary, we investigated the time course quantity of lysyl oxidase gene expression which perform significant role in the metabolism of collagen.
Wound healing model of rat were used, at the time of 1, 4, 7 days after making wounds through the whole skin , sample regenerated skins were collected and RNA was extracted. After Northern blotting, lysyl oxidase mRNA showed its peak at 4th day and normal level at 7th day. In situ hybridization showed positive grains on the fibroblasts existed in the periphery of granulation tissues. Increase of positive grains was corresponded to the result of Northern blotting.
We are now studying correlation of expression of types of collagen genes to above results of lysyl oxidase.