KISEP Rhinology Korean J Otolaryngol 2003;46:649-53 사람코점막과배양된코점막상피세포에서 15-Lipoxygenase 와 Cyclooxygenase-2 의발현 연세대학교의과대학이비인후과학교실, 1 두뇌한국 21 의과학사업단 2 김경수 1,2 윤주헌 1,2 김창훈 1 김선구 1 장정현 1 김지훈 1 Expression of 15-Lipoxygenase and Cyclooxygenase-2 in Human Nasal Mucosa and Cultured Human Nasal Epithelial Cells Kyung-Su Kim, MD 1,2, Joo-Heon Yoon, MD 1,2, Chang-Hoon Kim, MD 1, Sun Goo Kim, MD 1, Jung Hyun Chang, MD 1 and Ji Hoon Kim, MD 1 1 Department of Otorhinolaryngology, Yonsei University College of Medicine, Seoul; and 2 Brain Korea 21 Project for Medical Science, Seoul, Korea ABSTRACT Background and Objectives:The purpose of our study was to examine the expression of 15-lipoxygenase (15-LO) in human nasal mucosa and to investigate the change of expression of 15-LO and cyclooxygenase-2 (COX-2) as a function of mucociliary and squamous differentiation. Materials and Method:Human inferior turbinates were used and immunohistochemistry with 15-LO and COX-2 antibody was done. Passage-2 normal human nasal epithelial cell culture using air-liquid interface method was performed for 14 days and the cells were divided as retinoic acid (RA)-sufficient and RA-deficient group. Western blot analysis for 15-LO and COX-2 expression was performed on each group on 0, 7, and 14 days. Results:15-LO expression of mucociliated epithelium was noted in ciliated cells and basal cells, but was not found in goblet cells and secretory acini. In squamous epithelium, the expression of 15-LO was weaker than that in the mucociliated epithelium, but the expression of COX-2 showed no difference between them. In Western blot analysis, 15-LO expression was significantly higher in RA-sufficient culture than in RA-deficient culture and this expression was time-dependent. COX-2 expression was almost same level in RA-deficient culture, but its expression was significantly higher in RA-sufficient culture on 7 and 14 days than on zero day. Conclusion:15- LO and COX-2 may be related to differentiation and development of nasal epithelial cells. However, it is unclear whether this relationship is direct or indirect effect of 15-LO and COX-2. This question remains to be solved. (Korean J Otolaryngol 2003;46:649-53) KEY WORDS:Nasal mucosa 15-Lipoxygenase, arachidonate Synthase, prostaglandin Cell differentiation. - - - 649
사람코점막에서 15-LO 와 COX-2 의발현 - 650 - - - - - Korean J Otolaryngol 2003;46:649-53
김경수 외 전기영동은 8% SDS-polyacrylamide gel을 이용하였 게 발현되었으나, 배세포에서는 발현되지 않았다(Fig. 1A). 고 전기영동 후 nitrocellulose membrane에 전이시킨 다 분비선의 경우 점액 및 장액선 모두에서 발현되지 않았다 음 이 막을 10% milk in TBST로 4 에서 밤새 반응시킨 (Fig. 1C). 후 15-LO와 COX-2 항체로 실온에서 각각 4시간 동안 COX-2는 섬모상피세포의 세포질에서 주로 발현되었고 반응시켰다. TBST로 세척 후 horseradish peroxide 중합 일부 기저세포에서 발현되었으나 배세포에서는 발현되지 않 항체(Amersham Pharmacia Biotech, Buckinghamshire, 았고(Fig. 1B), 분비선에서도 발현되지 않았다(Fig. 1C). UK)로 1시간 처치한 다음 세척하였다. 이후 enhanced chemiluminescence(ecl kit, Amersham Pharmacia Biot- 일차 항체를 제외하고 염색한 음성 대조군의 경우 양성 반응이 관찰되지 않았다(Fig. 1D). ech)로 발색시킨 다음 autoradiography를 이용하여 15LO와 COX-2의 밴드를 각각 검출한 다음 이에 대해 Scion 점액섬모 상피조직과 편평 상피조직에서 15-LO와 COX- Image(Scion Co., Frederick, MD, USA)를 이용하여 각 2의 발현 비교 밴드의 세기를 반정량적으로 분석하였다. 위의 membrane 하비갑개의 전단부에서 편평 상피 부위와 중간부에서 점 을 deprobing 후 actin 항체(Santa Cruz Biotechnology 액섬모 상피조직 부위를 선택하였다. 각각에 대해 15-LO Inc., Santa Cruz, CA, USA)(1 500 농도)로 Western 와 COX-2 항체로 면역조직화학염색을 하여 분화에 따른 blot analysis하여 actin 밴드에 대한 세기를 구하였다. 15- 각 단백 발현의 차이를 알아보았다. LO와 COX-2 밴드의 세기를 actin 밴드의 세기에 대한 15-LO의 경우 점액섬모 상피에서 강하게 발현되었으나 비율(15-LO 또는 COX-2/actin)로 표시하여 강도를 비 편평 상피조직의 경우 발현이 매우 적거나 관찰되지 않아 교하였다. 점액섬모 상피조직보다 발현이 감소된 소견을 보였다(Fig. 2A and B). 결 과 COX-2의 경우 점액섬모 상피와 편평 상피조직간에 유사 하게 발현되어 유의한 차이를 보이지 않았다(Fig. 2C and D). 사람 정상 코점막에서 15-LO와 COX-2의 발현 사람 하비갑개 점막을 다클론성 15-LO와 COX-2 항체 로 면역조직화학염색을 하여 각 단백의 발현을 관찰하였다. 15-LO는 섬모상피세포와 기저세포의 세포질에서 강하 A B 사람 정상 코점막 상피세포에서 분화 양상 및 정도에 따 른 15-LO와 COX-2의 발현 사람 정상 코점막 상피세포를 섬모 상피세포로 분화시 C D Fig. 1. Expression of 15-lipoxygenase and cyclooxygenase-2 in human nasal mucosa. A 15-lipoxygenase expresses in the upper portion of cytoplasm of ciliated cells and basal cells, but not in goblet cells. B Cyclooxygenase-2 expresses mostly in upper portion of ciliated cells and partly in basal cells, but not in goblet cells. C In submucous glands, positive reactions to cyclooxygenase-2 and 15-lipoxygenase are not found. D Negative control in which the primary antibody was omitted. A B C D Fig. 2. Expression of 15-lipoxygenase and cyclooxygenase-2 in mucociliary and squamous epithelium. Expession of 15-lipoxygenase is more intense in mucociliated epithelium (A) than in squamous epithelium (B), but expression of cyclooxygenase is almost same level between mucociliated (C) and squamous (D) epithelium. 651
사람코점막에서 15-LO 와 COX-2 의발현 RA Actin Ratio - 652 15-LO 0 day 7 day 14 day COX-2 0 day 7 day 14 day 0.1 0.3 0.9 0.5 0.4 0.4 1.0 0.4 1.2 Fig. 3. Expression of 15-lipoxygenase 15-LO and cyclooxygenase-2 COX-2 according to culture duration in the presence or absence of retinoic acid RA. In Western blot analysis using normal human nasal epithelial cells grown in RA-sufficient RA or RA-deficient RA culture media, the values of 15-LO and COX-2 expression were marked as ratio using their corresponding actin level after the estimation of intensity of hybridization bands by densitometer. 15-LO expression is significantly higher in RA culture than in RA culture and the expression of 15-LO increases with culture duration in RA culture. COX-2 expression is almost same level in RA culture throughout culture duration. However, in RA culture, COX-2 expression increases on 7 and 14 days after confluence compared with that on the day of confluence, but there is no significant difference between 7 and 14 days. - Korean J Otolaryngol 2003;46:649-53
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