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Reconstructed skin provides advantages to single cell culture testing and leads to promising results as shown by different validation studies. In this study we investigated the efficacy of "KeraskinTM" compared with "EPI-200" and in vivo test. We conducted endpoint analysis including cell viability (MTT reduction, CCK-8) and IL-1 alpha release. Twenty chemical were investigated using keraskinTM model and compared with Epiderm model. Results of MTT assay and CCK-8 assay were not different between EPI-200 and KeraSkinTM, but IL-lα levels of some test items different between EPI-200 and KeraSkinTM. We concluded that this KeraSkinTM model is useful for alternative skin irritation test, although other tests should be conducted for many other chemicals in international validation study.
Zebrafish-based small molecule screens provide high-throughput drug discovery. Contrary to target- based approaches, this organism-based discovery provides information on toxicity, side effects, effectiveness, and pharmacokinetic profiles. In addition, it enables to find chemical modifiers of any biological process relevant to cancer including cell cycle, cell survival, apoptosis, and tumor suppression. To embody drug discovery using this model, it is necessary to generate a practical zebrafish model that harbors a specific derangement of signaling pathway listed above or transgenic activation of a target gene. Transgenic model allows generation of suitable model for drug screens. A strategy to append a biologic marker enables real-time observation of phenotypic reversal by chemical modifier and facilitates easy and rapid drug screening. In an effort to generate a zebrafish tumor model for platform of small molecular anticancer drug screening, oncogenic Kras was specifically overexpressed in exocrine pancreatic progenitor cells by using genetic recombineeringtechnology. Methods: Using BAC transgenes under regulation of ptf1a regulatory elements, we expressed either GFP alone or GFP fused to oncogenic Kras in developing zebrafish pancreas. To enable real-time observation of expressed transgene in a living zebrafish Kras transgene was generated as GFP-fused gene. Results: Transgene expression spatiotemporally recapitulated the ptf1a gene expression pattern during development. Following their initial specification, normal GFP-labeled pancreatic progenitor cells were observed to actively migrate away from the forming endodermal gut tube, and subsequently underwent characteristic exocrine differentiation. In contrast, pancreatic progenitor cells expressing oncogenic KRAS underwent normal specification and migration, but failed to differentiate. This block in differentiation resulted in the abnormal persistence of an undifferentiated progenitor pool, and was associated with the subsequent formation of invasive pancreatic cancer. Proportions of fish with detectable tumor-bearing transcutaneous fluorescence increased with advancing age of the fish, ie, 20% at 3 months, 41.7% at 6 months, and 66.7% at 9 months. The presence and size of detectable Tc-GFP were highly concordant with the presence of tumor and tumor burden, respectively. Though, most of the tumors showed acinar differentiation evidenced by weak expression of carboxypeptidase A and amylase, some tumors showed partly ductal or squamoid differentiation. With advance of lesion, tumor frequently revealed invasion and metastasis into bowel wall, liver, and ovary. These tumors exhibited several features in common with the human disease, including evidence of abnormal Hedgehog pathway activation. Conclusions: This zebrafish model of pancreatic tumor expressing surrogate markers demonstrates the feasibility of novel strategy to generate tumor models providing an effective platform for novel pancreatic cancer drug discovery. Additionally, these results provide a unique view of the tumor-initiating effects of oncogenic KRAS in a living vertebrate organism, and suggest that zebrafish models of pancreatic cancer may prove useful in advancing our understanding of the human disease.
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.
( Ji Young Moon ),( Jin Hee Lee ),( Eun Ho Maeng ),( Hak Soo Park ),( Min Kwon ),( Dong Hyouk Jang ),( Young Min Cho ),( Eun Ok Koh ),( Ha Jung Sung ),( Cheol Beom Park ) 한국동물실험대체법학회 2007 한국동물실험대체법학회 학술대회집 Vol.2007 No.1
Reconstructed skin give advantages to single cell culture testing system and leads to promising results as be shown in different validation studies. In this study we investigated the efficacy of MCTT Skin model compared with in vivo test. We conducted endpoint analysis including cell viability and IL-1 alpha release. Sodium lauryl sulfate(20%), Potassium hydoxide(5%), Heptanal, Methyl palmitate, 1,1,1-Trichloroethane, 2,4-Xylidine, 3,3`-Dithiodipropionic acid, 4-Amino-1,2,4-triazol and Benzyl chloroformate were selected for this comparison. Potassium hydoxide(5%) and Benzyl chloroformate were excluded from animal tests because of their pH. Sodium lauryl sulfate induced severe irritation, Heptanal induced moderate irritation. Methyl palmitate, 1,1,1-Trichloroethane induced mild irritation. 2,4-Xylidine, 3,3`-Dithiodipropionic acid, 4-Amino-1,2,4-triazole induced no irritation on rabbit skin irritation test. Except Methyl palmitate and 2,4-Xylidine, the results were same between MCTT Skin model and in vivo skin irritation test. According to other studies, the results of other skin model and in vivo test of Methyl palmitate and 2,4-Xylidine were shown different result each other. Thus, we concluded that this MCTT Skin model is useful for alternative skin irritation test, although other tests should be conducted for many other chemicals in international validation study.
The Irritection Assay System(R) was evaluated as alternative methods to the Draize skin irritation test (Draize test). This assay is based on the principle that chemicals that cause dermal irritation are known to induce alterations in the structure of keratin, collagen and other dermal proteins. The Irritection Assay System is an in vitro test that mimics these biochemical phenomena. This study was evaluated of 10 chemicals. Potassium hydroxide (5%) induced non-irritant and 3,3`-dithiodipropionic acid induced irritant, the results were not the same as Draize test. But eight of ten chemicals tested showed similar irritancy potential. Sodium lauryl sulfate (20%) induced irritant. 1,1,1-tricholoroethane, 1-bromopentane and cis-cyclooctene induced Non-irritant/ Irritant. Dimethyl sulfoxide, d-limonene, 2,4-xylidine, and 4-amino-1,2,4-tiazole induced Non-irritant on Irritection Assay System(R). We introduced that the Irritection Assay System(R) is easily alternative skin irritation evaluation method, in addition to planning to evaluate surfactant and cosmetic products.
The effect of the social enrichment program using mingle cages was investigated in hematological parameters of male cynomolgus monkeys. It is generally believed that the social enrichment program using mingle cages may offer the socialization and the prevention of isolation syndrome such as abnormal behavior. The total of 48 males and 42 females were housed in pairs homosexually with mingle cages reformed from individual cages for 9 weeks and we observed the clinical signs, body weight changes and hematological parameters. In the previous study, Han et al.,2007 observed clinical signs and body weight changes. 93.8 % of male cage mates and 100% of female cage mates were made successful pair-housing formation except 3 cases of male cage mates with serious injuries from fighting. And body weight gains were not changed between individual cage-housing and pair-housing for 9 weeks. However WBC count of male monkeys in individual cage was decreased compared with pair housing group after 8 and 21 weeks from pair housing. Significant increase in neutrophils and decrease in lymphocytes were observed in pair housing group. Thus, this finding indicate that housing condition may change hematological parameters. The present finding may serve as useful basic data for social enrichment program using mingle cage.
Prior to systemic investigation on intense husbandry environment-mediated stress outcome in pig or hen, we compared serum levels of epinephrine, norepinephrine, and cortisol in pigs or hens housed in intense husbandry confinement building or environmentally friendly husbandry facility. Pigs were accommodated under density of 3 per 3.3 ㎡and were kept on wet floors in the intense husbandry confinement building with fan-ventilation system. Meanwhile, the environmentally friendly husbandry facility with open ventilation system accommodated 2 pigs per 3.3 ㎡and pigs were kept on dry floors. Hens were housed under density of 3 per 0.1 ㎡in wire-partitioned battery cage at the intense husbandry confinement building with fan-ventilation system. Meanwhile, the environmentally friendly husbandry facility with open ventilation system accommodated 11 hens per 3.3 ㎡on flat ground. Five animals from each facility were randomly chosen and their peripheral blood was collected from jugular vein in pig or wing vein in hen. Levels of epinephrine, norepinephrine, and cortisol were determined using commercially available ELISA kit. Pigs housed in the intense husbandry confinement building demonstrated 10-fold and 6-fold lower level of epinephrine and norepinephrine, respectively, than those in the environmentally friendly husbandry facility. Furthermore, level of cortisol was significantly lowered in both pigs and hens housed in the intense husbandry confinement building, compared with that in the environmentally friendly husbandry facility. Overall our results suggest that pigs and hens housed in the intense husbandry confinement building are not adequately responding to extrinsic stress such as manual bleeding, eventually leading to vulnerability to various diseases.
( Hyoun Kyoung Lee ),( Kyoung Jin Noh ),( Seung Hyeok Seok ),( Min Won Baek ),( Hui Young Lee ),( Dong Jae Kim ),( Yi Rang Na ),( Sung Hoon Park ),( Dutta Noton Kumar ),( Byoung Hee Lee ),( Bae Hwan K 한국동물실험대체법학회 2007 한국동물실험대체법학회 학술대회집 Vol.2007 No.1
The necessity of using animals to test whether new chemicals and products are eye irritants has been questioned over the last 20 years. During this process, numerous non-animal methods have been proposed as reliable alternatives to the Draize test. The neutral red uptake assay is the in vitro method to test the eye irritation of chemicals and products. We need to validate the neutral red uptake assay as alternatives for the international harmonization. Therefore, we studied for the establishment of the neutral red uptake assay in Korea. In this study, we evaluated the eye irritation of five chemicals and three cosmetics by the Draize test and the neutral red uptake assay. The results of the neutral red uptake assay showed a correlation of r=−0.893(p=0.107) with our Draize score in two chemicals. And the results of the neutral red uptake assay showed correlation of r=0.892(p=0.017) with the Draize score obtained from the ECVAM (European Centre for the Validation of Alternative Methods) in additional three chemicals. All of three cosmetics were evaluated as non-irritants by our Draize test and the neutral red uptake assay. As a result, the neutral red uptake assay was established in Korea. For the international harmonization, the validation study has to be performed with more chemicals and products in more countries.
석승혁 ( Seung Hyeok Seok ),( Ju Hee Han ),( Min Won Baek ),( Hui Young Lee ),( Dong Jae Kim ),( Yi Rang Na ),( Hyun Kyoung Lee ),( Sung Hoon Park ),( Bae Hwan Kim ),( Kui Lea Park ),( Jong Kwon Lee ),( 한국동물실험대체법학회 2008 한국동물실험대체법학회 학술대회집 Vol.2008 No.1
The Draize eye irritation test has been changed to alternative methods based on 3 Rs (Replacement, Reduction, Refinement) of animal experiments. The bovine corneal opacity and permeability (BCOP) assay is well established as one of the alternative methods to the Draize test but the supply of bovine eyes from a slaughterhouse is not sufficient in actuality. The porcine corneal opacity and permeability (PCOP) assay have been proposed as a reliable alternative to the BCOP assay and the Draize test because porcine corneas have numerous advantages including structural similarities with human corneas and the stable capacity for a corneal supply. In this study, three cosmetics and seven chemicals that are already known as irritants at Draize score and the bovine corneal opacity (BCO) assay were tested by the PCOP assay for the first time in Korea. The porcine corneal opacity was measured by quantifying the ability of light to pass through a cornea using an opacitometer. The permeability was measured at 10 and 30 min time points after chemical exposure. The PCOP assay showed the high correlation with the Draize score and BCO assay. These results showed that the PCOP assay can be used as an alternative method for the BCO assay and the Draize test although it needs further studies about insoluble chemicals and others to increase reliability.