피부감작능 평가를 위한 BrdU-FCM 국소림프절시험법 기법 이전을 위한 제 조건

여경욱 ( Kyeong Uk Yeo ),김지연 ( Ji Youn Kim ),황소련 ( So Ryeon Hwang ),임경민 ( Kyung Min Lim ),정경미 ( Kyung Mi Jung ),장원희 ( Won Hee Jang ),허용 ( Yong Heo ) 한국동물실험대체법학회 2012 동물실험대체법학회지 Vol.6 No.1

연구논문 : THP-1 수지상세포주와 HaCaT 각질세포주 공배양을 통한 피부감작성 평가 대체시험법 개발

여경욱 ( Kyeong Uk Yeo ),차원석 ( Won Seok Cha ),연동은 ( Dong Eun Yeon ),최성희 ( Sung Hee Choi ),이지연 ( Ji Youn Lee ),신경민 ( Kyeong Min Shin ),조지훈 ( Ji Hoon Jo ),허용 ( Yong Heo ) 한국동물실험대체법학회 2013 동물실험대체법학회지 Vol.7 No.1

본 연구진은 THP-1 수지상세포주에서 생성되는 MIP-1β 싸이토카인이 피부 비감작성 단순 자극성 물질을 첨가하였을 때 비해 피부감작물질을 첨가하였을때 유의하게 높은 수준으로 생성됨을 보고하면서 MIP-1β가 피부감작능 평가 대체시험법에서 biomarker로 개발될 수 있음을 제시하였다 (안 등, 2008; Lim et al., 2008). 또한 HaCaT 각질세포주에서 생성되는 IL-6 수준 증가가 피부감작물질과 비감작물질을 구분 해줄 수 있는 친염증성 싸이토카인 중 하나임을 보고하고 HaCaT 세포주를 이용한 피부감작능 대체시험법 개발 가능성을 제시하였다 (김 등, 2011 & 2012). 실제 in vivo에서 각질세포는 피부 Langerhans 탐식 세포와 상호작용을 통하여 접촉성 알레르기성 피부염의 진행을 유도하는 것으로 알려져 있다 (Kimber et al., 2004). THP-1 세포주는 원래 단핵구성 백혈구 암세포 (monocytic leukemia cell)로 알려져 있었으나 지속적인 연구를 통하여 수지상세포의 특성을 갖고 있음이 보고되었고 현재 이 세포주를 이용한 h-CLAT 시험법은 국제적인 검증과정에 있다 (Ashikaga et al., 2010). 본 연구는 피부감작능 평가시 각질세포와 수지상세포의 상호 작용이 기전상 중요하고 이를 in vitro에서 적절하게 재현하여 보다 정확도가 높은 피부감작성평가 대체시험법을 개발하고자 시도된 최초의 탐색연구이다. THP-1 세포주에서 생성되는 MIP-1β와 HaCaT 세포주에서 주로 생성되는 IL-6, IL-8 수준을 세포배양액에서 측정하였고, 용매 대조 물질을 첨가한 군에 비해 몇 가지 화학물질을 첨가한 경우에 어느 정도 이들 싸이토카인이 생성되었는지를 자극지수로 하여 계산하였다. 본 연구 결과 24-well plate의 바닥에서 배양한 THP-1 세포에서 생성되는 MIP-1β가 HaCaT 세포가 존재하는 culture plate insert 안에서 채취한 배양액에서도 유사한 수준으로 측정되었는데 이는 pore size 0.4 ㎛인 culture plate insert를 통하여 싸이토카인이 이동하고 있음을 알 수 있었다 (Table 1). 또한 HaCaT 세포 배양시 통상적으로 사용되는 DMEM 배지보다도 RPMI 배지의 경우가 IL-6, IL-8 싸이토카인 수준이 높았다는 점에서 추후 공배양을 이용한 대체시험법 개발시 RPMI 배지를 사용할 것이 권장된다. 본 연구의 핵심 결과는 MIP-1β 생성 수준이 DNCB와 HCA 모두 1X 농도에서는 LLNA에서 피부감작물질로 판정하는 3이상의 자극지수를 나타내면서 비감작성 물질 lactic acid 첨가시 생성 수준보다 유의하게 높았다는 것이다 (Table 2). 과거 본 연구진의 THP-1 세포주 단일 배양시 MIP-1β 생성 수준이 1X DNCB에서 246.0±68.5%, 0.1X HCA에서 167.0±3.7%로 최고였던 결과와 (Lim et al., 2008) 비교하면 이번 연구에서 1X DNCB 및 1XHCA 각각 308±147%, 345±13%로 높았던 점은 피부감작물질 노출시 THP-1 세포의 활성화가 각질세포와 공배양을 통하여 증진되는 측면이 있을 가능성을 제시하는 것으로 판단된다. 본 공배양체계에서 DNCB, HCA 첨가시 IL-6 생성 수준은 본 연구진의 최근 연구에서 (김 등, 2012) HaCaT 세포주 단일 배양시 얻어졌던 결과 (0.5X DNCB: 158±20%, 0.5X HCA: 156±23%)에는 미치지 못하였다. 비록 초기 탐색 연구이지만 본 연구를 통하여 THP-1 수지상세포주와 HaCaT 각질세포주를 공배양하였을 때 MIP-1β 생성 수준을 평가함으로써 피부감작능 여부를 예측할 수 있는 가능성이 높음을 알 수 있었다. 본 시험 결과는 보다 많은 시험물질을 사용하여 보다 체계적인 연구를 통하여 확인할 필요성이 높다고 생각된다. Co-culture system of THP-1 dendritic cell line and HaCaT keratinocyte cell line was developed for its usefulness in sorting out chemicals with skin allergic potential. THP-1 cells were plated onto wells of 24-well culture plate followed by placement of culture plate insert to each well. Thereafter HaCaT cells were added into the culture plate inserts. Levels of macrophage inflammatory protein-1β (MIP-1β), interleukin-6 (IL-6), and interleukin-8 (IL-8) production were determined using culture supernatants from HaCaT cell or THP-1 cell culture area. The optimal culture duration was 48 hour among 24, 48, and 72 hour for production of those cytokines. In addition, complete RPMI medium was better than complete DMEM medium. Co-culture of THP-1 and HaCaT cells was maintained in the presence or absence of 4 doses, 0.01X, 0.1X, 0.5X, or 1X CV75 (75% cell viability concentration) of 2 sensitizing chemicals (dinitrochlorobenzene, hexyl cinnamic aldehyde) and an irritant chemical (lactic acid) for 48 hour. Stimulation index (SI) was calculated through dividing each cytokine amount at each chemical concentration by vehicle control`s cytokine amount. When SI 3 is considered as a threshold for categorizing chemicals into skin-sensitizer, dinitrochlorobenzene and hexyl cinnamic aldehyde were positive for MIP-1β, but no chemicals were above SI 3 for both IL-6 and IL-8. Further evaluations for other various cytokines and cytokine determination using cell lysates should be performed for examining usefulness of the co-culture system as a skin sensitizing alternative method.

THP-1 수지상세포주와 HaCaT 각질세포주 공배양을 통한 피부감작성 평가 대체시험법 개발

여경욱 ( Kyeong Uk Yeo ),차원석 ( Won Seok Cha ),연동은 ( Dong Eun Yeon ),최성희 ( Sung Hee Choi ),이지연 ( Ji Youn Lee ),신경민 ( Kyeong Min Shin ),조지훈 ( Ji Hoon Jo ),허용 ( Yong Heo ) 한국동물실험대체법학회 2013 동물실험대체법학회지 Vol.7 No.1

본 연구진은 THP-1 수지상세포주에서 생성되는 MIP-1β 싸이토카인이 피부 비감작성 단순 자극성 물질을 첨가하였을 때 비해 피부감작물질을 첨가하였을때 유의하게 높은 수준으로 생성됨을 보고하면서 MIP-1β가 피부감작능 평가 대체시험법에서 biomarker로 개발될 수 있음을 제시하였다 (안 등, 2008; Lim et al., 2008). 또한 HaCaT 각질세포주에서 생성되는 IL-6 수준 증가가 피부감작물질과 비감작물질을 구분 해줄 수 있는 친염증성 싸이토카인 중 하나임을 보고하고 HaCaT 세포주를 이용한 피부감작능 대체시험법 개발 가능성을 제시하였다 (김 등, 2011 & 2012). 실제 in vivo에서 각질세포는 피부 Langerhans 탐식 세포와 상호작용을 통하여 접촉성 알레르기성 피부염의 진행을 유도하는 것으로 알려져 있다 (Kimber et al., 2004). THP-1 세포주는 원래 단핵구성 백혈구 암세포 (monocytic leukemia cell)로 알려져 있었으나 지속적인 연구를 통하여 수지상세포의 특성을 갖고 있음이 보고되었고 현재 이 세포주를 이용한 h-CLAT 시험법은 국제적인 검증과정에 있다 (Ashikaga et al., 2010). 본 연구는 피부감작능 평가시 각질세포와 수지상세포의 상호 작용이 기전상 중요하고 이를 in vitro에서 적절하게 재현하여 보다 정확도가 높은 피부감작성평가 대체시험법을 개발하고자 시도된 최초의 탐색연구이다. THP-1 세포주에서 생성되는 MIP-1β와 HaCaT 세포주에서 주로 생성되는 IL-6, IL-8 수준을 세포배양액에서 측정하였고, 용매 대조 물질을 첨가한 군에 비해 몇 가지 화학물질을 첨가한 경우에 어느 정도 이들 싸이토카인이 생성되었는지를 자극지수로 하여 계산하였다. 본 연구 결과 24-well plate의 바닥에서 배양한 THP-1 세포에서 생성되는 MIP-1β가 HaCaT 세포가 존재하는 culture plate insert 안에서 채취한 배양액에서도 유사한 수준으로 측정되었는데 이는 pore size 0.4 ㎛인 culture plate insert를 통하여 싸이토카인이 이동하고 있음을 알 수 있었다 (Table 1). 또한 HaCaT 세포 배양시 통상적으로 사용되는 DMEM 배지보다도 RPMI 배지의 경우가 IL-6, IL-8 싸이토카인 수준이 높았다는 점에서 추후 공배양을 이용한 대체시험법 개발시 RPMI 배지를 사용할 것이 권장된다. 본 연구의 핵심 결과는 MIP-1β 생성 수준이 DNCB와 HCA 모두 1X 농도에서는 LLNA에서 피부감작물질로 판정하는 3이상의 자극지수를 나타내면서 비감작성 물질 lactic acid 첨가시 생성 수준보다 유의하게 높았다는 것이다 (Table 2). 과거 본 연구진의 THP-1 세포주 단일 배양시 MIP-1β 생성 수준이 1X DNCB에서 246.0±68.5%, 0.1X HCA에서 167.0±3.7%로 최고였던 결과와 (Lim et al., 2008) 비교하면 이번 연구에서 1X DNCB 및 1XHCA 각각 308±147%, 345±13%로 높았던 점은 피부감작물질 노출시 THP-1 세포의 활성화가 각질세포와 공배양을 통하여 증진되는 측면이 있을 가능성을 제시하는 것으로 판단된다. 본 공배양체계에서 DNCB, HCA 첨가시 IL-6 생성 수준은 본 연구진의 최근 연구에서 (김 등, 2012) HaCaT 세포주 단일 배양시 얻어졌던 결과 (0.5X DNCB: 158±20%, 0.5X HCA: 156±23%)에는 미치지 못하였다. 비록 초기 탐색 연구이지만 본 연구를 통하여 THP-1 수지상세포주와 HaCaT 각질세포주를 공배양하였을 때 MIP-1β 생성 수준을 평가함으로써 피부감작능 여부를 예측할 수 있는 가능성이 높음을 알 수 있었다. 본 시험 결과는 보다 많은 시험물질을 사용하여 보다 체계적인 연구를 통하여 확인할 필요성이 높다고 생각된다. Co-culture system of THP-1 dendritic cell line and HaCaT keratinocyte cell line was developed for its usefulness in sorting out chemicals with skin allergic potential. THP-1 cells were plated onto wells of 24-well culture plate followed by placement of culture plate insert to each well. Thereafter HaCaT cells were added into the culture plate inserts. Levels of macrophage inflammatory protein-1β (MIP-1β), interleukin-6 (IL-6), and interleukin-8 (IL-8) production were determined using culture supernatants from HaCaT cell or THP-1 cell culture area. The optimal culture duration was 48 hour among 24, 48, and 72 hour for production of those cytokines. In addition, complete RPMI medium was better than complete DMEM medium. Co-culture of THP-1 and HaCaT cells was maintained in the presence or absence of 4 doses, 0.01X, 0.1X, 0.5X, or 1X CV75 (75% cell viability concentration) of 2 sensitizing chemicals (dinitrochlorobenzene, hexyl cinnamic aldehyde) and an irritant chemical (lactic acid) for 48 hour. Stimulation index (SI) was calculated through dividing each cytokine amount at each chemical concentration by vehicle control`s cytokine amount. When SI 3 is considered as a threshold for categorizing chemicals into skin-sensitizer, dinitrochlorobenzene and hexyl cinnamic aldehyde were positive for MIP-1β, but no chemicals were above SI 3 for both IL-6 and IL-8. Further evaluations for other various cytokines and cytokine determination using cell lysates should be performed for examining usefulness of the co-culture system as a skin sensitizing alternative method.

피부감작능 평가를 위한 BrdU-FCM 국소림프절시험법 기법 이전을 위한 제 조건

여경욱 ( Kyeong Uk Yeo ),김지연 ( Ji Youn Kim ),황소련 ( So Ryeon Hwang ),임경민 ( Kyung Min Lim ),정경미 ( Kyung Mi Jung ),장원희 ( Won Hee Jang ),허용 ( Yong Heo ) 한국동물실험대체법학회 2012 동물실험대체법학회지 Vol.6 No.1

Local lymph node assay (LLNA) using mice has been adopted as an alternative model for testing skin sensitization to chemicals. Modification of LLNA was recently reported through combination of BrdU incorporation and flow cytometric analysis, which was designated as LLNA:BrdU-FCM. We introduce how LLNA:BrdU-FCM could be successfully established in a naive laboratory where the method was never experienced. Nine week old female BALB/c mice, 5 mice per group, were used and grouped into non-treatment, BrdU only-treatment, acetone-olive oil (AOO) vehicle-treatment, and 5, 10, 25% α-hexylcinamaldehyde (HCA) positive control. BrdU solution was injected intraperitoneally one day after three consecutive application of AOO or HCA on dorsum area of both ears. Auricular lymph node was collected from both ears one day after the BrdU injection. Thereafter flow cytometric staining procedures were followed. Various parameters related with calculation of stimulation index were analyzed including number of lymphocytes, BrdU incorporation%, and number of BrdU incorporated lymphocytes. The critical procedures for successful establishment were as followings; quality control of every quantitative apparatus, appropriate application of test compounds on dorsum of mouse ear, precise injection of BrdU, accurate preparation of single cell suspension and counting of lymph node cells, and experienced operation of flow cytometer. Face-to-face training from an experienced lead laboratory and preparation of very detailed standard operational procedures could play the most important role for the establishment. Based on Performance Standards for Assessment of Proposed Similar or Modified Test Methods by OECD, the LLNA:BrdU-FCM could be a good alternative method for screening skin sensitizers.

각질세포주로부터 Interleukin-6 생성 수준에 따른 피부감작물질 검색 유용성 분석

김소남 ( So Nam Kim ),여경욱 ( Kyeong Uk Yeo ),김형아 ( Hyoung Ah Kim ),허용 ( Yong Heo ) 한국동물실험대체법학회 2012 동물실험대체법학회지 Vol.6 No.1

HaCaT cell, a human keratinocyte cell line, was evaluated for its usefulness in sorting out chemicals with skin allergic potential. Interleukin-6 production was determined in HaCaT cell culture supernatants and cell lysates. HaCaT cells were cultured in the presence or absence of 4 doses, 0.01X, 0.1X, 0.5X, or 1X CV75 (75% cell viability concentration) of 3 sensitizing chemicals (dinitrochlorobenzene, hexyl cinnamic aldehyde, cobalt chloride) and 2 irritant chemicals (lactic acid, isopropanol) for 48 and 72 hours. IL-6 stimulation index (SI) was calculated through dividing IL-6 amount at each chemical concentration by IL-6 amount for vehicle control. When SI exceeding 3 is considered a threshold for categorizing into skin-sensitizer, dinitrochlorobenzene and hexyl cinnamic aldehyde were positive since SI was 7.36 and 3.03 for dinitrochlorobenzene and hexyl cinnamic aldehyde, respectively, for cell lysates obtained from 48 hour cultured-HaCaT cells. Two non-sensitizing chemicals, lactic acid and isopropanol, demonstrated SI value below 3, indicating negative to skin sensitizing potential. These results indicate that IL-6 production could be a biomarker for classification of chemicals as sensitizers or non-sensitizers.

연구논문 : 정각질세포주의 피부감작물질 검색 관련 효용성 분석

김소남 ( So Nam Kim ),여경욱 ( Kyeong Uk Yeo ),황소련 ( So Ryeon Hwang ),조지훈 ( Ji Hoon Jo ),손수정 ( Soo Jung Sohn ),염영나 ( Young Na Yum ),이용경 ( Yong Kyung Lee ),허용 ( Yong Heo ) 한국동물실험대체법학회 2011 동물실험대체법학회지 Vol.5 No.1

Alternative methods using various human or animal cell lines are under development to classify chemical ingredients to skin sensitizing or irritant chemicals. Keratinocyte cell line could be a good target cell, in that the cells are the first contact point for chemical absorption through skin, and immune reactions including inflammation are occurred through interactions among keratinocyte, Langerhans` cell, and T cells. This study introduced current research activities on development of alternative methods using keratinocyte cell lines for identifying skin sensitizers. In addition, a good laboratory practice was introduced to perform serial passage culture of HaCaT human keratinocyte cell line. Furthermore, optimal cell density and culture duration were demonstrated to determine both intracellular and extracellular levels of IL-1α, IL-6, IL-8, and IL-18 from HaCaT cells.

정각질세포주의 피부감작물질 검색 관련 효용성 분석

김소남 ( So Nam Kim ),여경욱 ( Kyeong Uk Yeo ),황소련 ( So Ryeon Hwang ),조지훈 ( Ji Hoon Jo ),손수정 ( Soo Jung Sohn ),염영나 ( Young Na Yum ),이용경 ( Yong Kyung Lee ),허용 ( Yong Heo ) 한국동물실험대체법학회 2011 동물실험대체법학회지 Vol.5 No.1

Alternative methods using various human or animal cell lines are under development to classify chemical ingredients to skin sensitizing or irritant chemicals. Keratinocyte cell line could be a good target cell, in that the cells are the first contact point for chemical absorption through skin, and immune reactions including inflammation are occurred through interactions among keratinocyte, Langerhans` cell, and T cells. This study introduced current research activities on development of alternative methods using keratinocyte cell lines for identifying skin sensitizers. In addition, a good laboratory practice was introduced to perform serial passage culture of HaCaT human keratinocyte cell line. Furthermore, optimal cell density and culture duration were demonstrated to determine both intracellular and extracellular levels of IL-1α, IL-6, IL-8, and IL-18 from HaCaT cells.

각질세포주로부터 Interleukin-6 생성 수준에 따른 피부감작물질 검색 유용성 분석

김소남 ( So Nam Kim ),여경욱 ( Kyeong Uk Yeo ),김형아 ( Hyoung Ah Kim ),허용 ( Yong Heo ) 한국동물실험대체법학회 2012 동물실험대체법학회지 Vol.6 No.1

자폐증 유사증상 발현 마우스의 점액 면역에 대한 연구

황소련,조지훈,신경민,장윤영,김지연,여경욱,김형아,허용,Hwang, So Ryeon,Jo, Ji Hoon,Shin, Kyeong Min,Jang, Yun Young,Kim, Ji Youn,Yeo, Kyeong Uk,Kim, Hyoung Ah,Heo, Yong 한국환경보건학회 2012 한국환경보건학회지 Vol.38 No.6

Objectives: This study was undertaken in order to evaluate a potential mechanism involved in gastro-intestinal problems observed in autistic subjects and uses an animal model of autism investigation. Methods: BTBR T+tf/J, a mouse strain with typical socio-behavioral characteristics of autistic subjects and FVB mice with highly social behaviors as the control strain were used. Both genders of mice aged three weeks and six months were used from four separate litters for each strain. Serum was prepared following cardiac puncture, and mesenteric lymph nodes were collected for in vitro stimulation and enumeration of major immune cell proportion. Results: The level of serum IgA was significantly enhanced in six-month-old BTBR mice compared with three-week-old BTBR, which was not observed with the FVB control mice. The serum IgE level was also higher among BTBR mice than among age-sex matched FVB mice, respectively. Considering the ratio of interleukin-4 vs interferon-gamma production from mesenteric lymph node T cells, skewedness toward type-2 reactivities was observed. In addition, the proportion of B cells in mesenteric lymph nodes was significantly higher in BTBR mice than in FVB mice. Conclusion: Upregulation of mucosal immunity related with enhanced type-2 immune reactivity observed in BTBR mice could be involved with the etiology of gastro-intestinal abnormalities in autism.

경기 일부 지역 양돈 축산인들의 호흡기 알레르기 관련 면역학적 지표 분석

김지연 ( Ji Youn Kim ),김광호 ( Kwang Ho Kim ),황소련 ( So Ryeon Hwang ),여경욱 ( Kyeong Uk Yeo ),김형아 ( Hyoung Ah Kim ),허용 ( Yong Heo ) 한국산업위생학회 2012 한국산업보건학회지 Vol.22 No.4

Objectives: Animal husbandry workers could be exposed to various work hazards including toxic gases, chemicals such as pesticides or organic dust. Immunological evaluation focusing on respiratory allergic hypersensitivity occurrence was undertaken for swine farm workers as a part of the study on immunologic status of dairy barn, swine confinement, and poultry farm workers. Materials aanndd Methods: Peripheral bloods were collected from 25 workers at the year of 2001 and 12 workers at the year of 2012 from swine farms located at Gyeonggi province, Korea. Seven adults not involved with animal husbandry were recruited at the year of 2001 from the same residential area as the swine farm workers`. Level of plasma IgE and 20 respiratory allergen-specific IgE were evaluated using commercially available ELISA kit. Results: Plasma IgE level was approximately five-fold higher in the swine farm workers regardless of the sampling year than the control subjects. Plant allergens from outdoor environments such as golden rod, pigweed, Russian thistle, or ragweed were the major allergens with positive reaction(allergen specific IgE≥0.7 IU/mL) for the swine farm workers at 2001 year. Meanwhile, house dust mite(Dermatophagoides farinae, D. pteronyssinus) and cockroach, typical indoor allergens in Korea, were the major respiratory allergens for the swine farm workers at 2012 year. Conclusions: Overall, even though our results are primitive, the results suggest that immunological function of swine farm workers could be modulated toward type-2 reactivity.