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Journal of Korean Cleft Palate-Craniofacial Association 2003;4(1):87-93.
Localization of Cyclooxygenase Isozymes in Dermal Wound Healing in Mouse.
Jun Hyeok Koh, Kwang Seog Kim, Dae Young Kim, Sam Yong Lee, Bek Hyun Cho, Chun Sang Bae
1Department of Plastic and Reconstructive Surgery, Chonnam National University Medical School, Kwangju, Korea. ir01212@hanmail. net
2Department of Anatomy, Chonnam National University Medical School, Kwangju, Korea.
Abstract
Cyclooxygenase(COX)-1 and COX-2 expression in dermal wound healing of mouse was detected by immunohistochemistry and Western blot analysis. In order to gain more information on the functional importance of COX-1 and COX-2 in dermal wound healing, we analysed COX-1 and COX-2 protein levels using the Western blotting technique. In addition, we used immunohistochemistry to determine the cellular localization of the protein products. The collected skins were rapidly frozen and kept at -70degrees Cuntil assayed. Each frozen skin was lysed with 0.5 ml of ice-cold solution. Large tissue debris and nuclear fragments were removed by two low-speed centrifugations and the resulting supernatant fraction was used for blots. The skin extracts were stored below -20degrees Cfor further experiments. By Western blotting, compared to the activity of COX-2 in normal skin, its activity was increased at days 1, 4, 8, and 12 and was maximal at 1 day after incisional wound of mouse skin whereas COX-1 was barely detectable. In normal skin, COX-1 immunostaining was observed among the basal cells of epidermis whereas COX-2 immunostaining was detected in the more differentiated, suprabasal keratinocytes. At post-incision 1-4 days, COX-2 staining was particularly prominent in the inflammatory cells, and at day 8, many macrophage-like cells were stained positively. COX-2 immunoreactive fibroblast, macrophage-like cells, and newly formed vascular endothelial cells were increased in number at 12 days after incision. These data suggest that COX-2 is constitutively expressed, just as is COX-1, in epidermis and is associated with keratinocyte differentiation. In addition, these findings support the well-established role for COX-2, the prostaglandins that they generate, as mediators of inflammatory response.
Keywords: Cyclooxygenase (COX)-1; Cyclooxygenase (COX)-2; Immunohistochemistry; Western blot analysis; Localization
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