• KSBB Journal
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• 제21권3호
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• pp.171-174
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• 2006

형광을 이용한 암 진단을 위해 배양된 정상 및 암세포주에 광민감제인 5-ALA를 투여하고 세포 내 외에서 생성된 Protoporphyrin IX(PpIX)의 형광을 측정하여 5-ALA 투여의 최적농도를 조사하였다. 정상 간세포주(Chang) 및 자궁경부암 세포주(HeLa)에 5-ALA를 농도별로 투여하여 ALA에 의해 유도된 PpIX의 생성을 확인하고, MTT assay로 세포생존율을 측정하였다. 배양된 cell에 5-ALA를 투여한 후 24시간 동안 배양함으로써 생성되는 PpIX의 양은 형광의 강도로 측정하였다. HeLa 세포주에 대한 5-ALA의 최적농도는 $50{\mu}g/ml$이며, 이 때의 형광(emission) 스펙트럼은 여기 파장이 410 nm일 때 602.3 nm, 659.9 nm에서 형광 봉우리가 관찰되었다. PpIX의 형광 강도를 측정한 결과, PpIX는 정상세포에서는 낮은 농도로 축적이 되는 반면에 암세포에서 더 높은 농도로 축적되었으며, 세포 외보다는 세포 내에서 더 높은 농도로 축적됨을 알 수 있었다.

• Asian-Australasian Journal of Animal Sciences
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• 제23권4호
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• pp.430-436
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• 2010

The objective of the present studies was to develop and validate a system for isolation, purification and extended culture of pigment-producing cells in alpaca skin (melanocytes) responsible for coat color and to determine the effect of alpha melanocyte stimulating hormone treatment on mRNA expression for the melanocortin 1 receptor, a key gene involved in coat color regulation in other species. Skin punch biopsies were harvested from the dorsal region of 1-3 yr old alpacas and three different enzyme digestion methods were evaluated for effects on yield of viable cells and attachment in vitro. Greatest cell yields and attachment were obtained following dispersion with dispase II relative to trypsin and trypsin-EDTA treatment. Culture of cells in medium supplemented with basic fibroblast growth factor, bovine pituitary extract, hydrocortisone, insulin, 12-O-tetradecanolphorbol-13-acetate and cholera toxin yielded highly pure populations of melanocytes by passage 3 as confirmed by detection of tyrosinase activity and immunocytochemical localization of melanocyte markers including tyrosinase, S-100 and micropthalmia-associated transcription factor. Abundance of mRNA for tyrosinase, a key enzyme in melanocyte pigment production, was maintained through 10 passages showing preservation of melanocyte phenotypic characteristics with extended culture. To determine hormonal responsiveness of cultured melanocytes and investigate regulation of melanocortin 1 receptor expression, cultured melanocytes were treated with increasing concentrations of ${\alpha}$-melanocyte stimulating hormone. Treatment with ${\alpha}$-melanocyte stimulating hormone increased melanocortin receptor 1 mRNA in a dose dependent fashion. The results demonstrated culture of pure populations of alpaca melanocytes to 10 passages and illustrate the potential utility of such cells for studies of intrinsic and extrinsic regulation of genes controlling pigmentation and coat color in fiber-producing species.

• 농약과학회지
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• 제12권4호
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• pp.342-350
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• 2008

최근 내분비장애 추정물질의 분류를 위해 많은 시험법이 연구되고 있으며 미국 EPA와 OECD에서는 시험법을 설정하려고 노력하고 있다. 추후 기등록농약에 대한 자료요구 또는 신규 등록농약 적용 등록기준의 추가 등을 고려하여 내분비계장애 추정물질 관련 OECD와 EPA에서 권장하는 시험법을 확립하고자 본 연구를 수행하였다. 시험약제를 30일간 경구 투여하여 조사한 결과, metribuzin 투여 수컷에서 부고환, 전립선, 정낭의 중량이 증가하였고 갑상선에서는 유의한 중량변화가 나타나지 않았다. 암컷에서는 갑상선의 중량 감소가 나타난 반면에 생식장기 중량에는 유의적인 변화가 없었다. Metribuzin 투여수컷에서 testosterone이 100 mg/kg/day 처리수준에서 감소하였고 FT4가 50, 100 mg/kg 수준에서 증가하였다. 암컷에서는 T3가 50, 100 mg/kg/day 수준에서 증가하여 갑상선 호르몬에 영향이 나타나는 것을 볼 수 있었다. 시험세포를 이용한 시험결과, 시험약제를 1 nM에서 1,000 nM까지 처리하였을 때 음성대조군과 비교할 때 metribuzin은 106-122%의 영향을 나타내어 세포이용시험에서는 metribuzin이 갑상선 호르몬성 영향을 보인 것으로 나타났다. 항갑상선 호르몬성 영향 시험에서는 시험약제 100 nM과 T4의 혼합 처리시 metribuzin은 양성 대조군과 비교하여 감소하여 항갑상선 호르몬성 영향을 나타내었다. 본 시험을 통하여 OECD TG 407과 EDSTAC에서 권고하는 pubertal assay와 수의과학 검역원에서 제조한 HeLaTRE cell을 이용한 in vitro 시험이 갑상선 호르몬성 영향 검색 시험으로 활용될 가능성이 있는 것으로 사료되었다.

• 한국연초학회지
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• 제26권2호
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• pp.152-158
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• 2004

Among short-term in vitro genotoxicity assays, micronucleus assays are rapid, inexpensive, and less labor-intensive system. We have undertaken a comparative study of sensitivity of cigarette smoke condensate(CSC) by general micronucleus(MN) assay and cytokinesis-block micronucleus(CBMN) assay. In this study, V79 Chinese hamster cells were employed to evaluate and compare the genotoxicity of CSC of Kentucky Reference Cigarette 2R4F by 2 kinds of in vitro MN assay methods. To determine the optimum concentration of cytochalasin B(CYB) to obtain the maximal number of binucleated cells for CBMN assay, triplicate cultures of growing cells were treated with CYB for 15 h. CYB treatments caused a concentration-dependent increase in cytotoxicity($1\~4{\mu}g/mL$) and proportion($0.25\~1\;{\mu}g/mL$) of binucleated cells. These data suggested that 1 ug/mL of CYB is as an optimum dose for CBMN assay in binucleated V79 cells. Short treatment(4 h) of CSC induced a micronucleated cells with a concentration-dependent response in the presence or absence of CYB, but CSC-induced MNs were weakened when S9 was present. Long treatments(19 h) of CSC also induced a significant increase MN formation with a concentration-dependent response. At a concentration of 75 ${mu}g/mL$, the MN cell frequencies of general MN assay and CBMN assay were $6.5\%\;and\;11.7\%$, respectively. Linear regression analysis revealed a good correlation in CBMN assay between a concentration of CSC and MN cell frequency. All these data indicated that CBMN assay is more sensitive to the induction CSC-induced MN than general MN assay.

• 한국발생생물학회지:발생과생식
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• 제13권4호
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• pp.217-226
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• 2009

본 연구는 내분비 장애물질의 검출을 위하여 간세포의 단층 형성, 생존 및 기능에 미치는 어류 혈청의 영향에 대해 검토하였다. 한국산 메기의 간세포는 자신의 혈청 및 뱀장어, 틸라피아 등 타어종의 혈청에 의해 부착 및 단층이 형성되었으나, FBS는 메기 간세포의 단층을 형성시키지 못했다. 0.5에서 3%의 어류 혈청으로 메기 간세포의 단층을 형성 시킬 수 있는데, 이것은 FBS(5~20%) 사용의 1/10 이하로 적은 양이며, 어류 혈청이 FBS를 대체할 수 있고, FBS보다 간세포의 형태 및 기능 유지에 효과적인 것으로 나타났다. 어류 혈청이 첨가된 배양액에서 메기 간세포는 적어도 10일 이상 단층 형성을 유지할 수 있어, 내분비 장애물질 연구에 이용될 수 있을 것이다. 결론적으로 본 연구에서 개발된 어류 혈청을 사용한 메기 간세포 배양시스템과 효소면역측정법(ELISA)은 bisphenol A 등의 내분비 장애물질의 검출 및 연구를 위한 유용한 도구로서 이용될 수 있다고 생각된다.

• 농약과학회지
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• 제4권2호
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• pp.1-10
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• 2000

국내 살균제 저항성 연구는 아직 초보적 수준으로 대부분의 연구가 실내에서의 살균제 저항성 균주의 검출보고에 국한하고 있다. 향후 살균제 저항성연구는 포장에서 대상 병원균 집단의 살균제에 대한 감수성 분포를 근거로 하여 그 살균제에 대한 저항성을 판별하는 기준 농도를 설정한 후 병원균주의 저항성 여부가 판정되어야 한다. 국내의 대부분의 연구가 이점을 간과하고 있기 때문에 연구자간에 저항성 기준에 차이가 있어 병원균 집단의 약제에 대한 감수성 변화를 추적하거나 해석하는데 많은 문제점이 있었다. 실내시험에서 검출된 살균제 저항성은 포장에서 감수성 및 저항성 균을 대상으로 방제효과를 조사한 후 그 저항성을 실증할 수 있어야 한다. 살균제 저항성은 실제로 농가포장에서의 약재 방제효율의 저하와 연관되어야 비로서 실용적인 의미를 갖기 때문이다. 저항성 균주에 대한 기생적 적응력의 조사는 병원균집단내의 저항성 균주들의 생존력이나 안정성을 검정하기 위하여 반드시 필요하다. 또한 살균제 저항성에 대한 장.단기적 대책을 강구하기 위하여 병원균 집단의 특정 살균제에 대한 감수성의 변화가 그 살균제를 사용하고 있거나 사용한 적이 없는 포장에서 주의 깊게 조사되어야만 한다. 이러한 연구들은 국내 살균제 저항성 문제를 해결하기 위한 기본적인 정보를 제공한다는 면에서 매우 시급한 연구과제가 되고 있다.

• Archives of Pharmacal Research
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• 제19권4호
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• pp.251-257
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• 1996

The detection of many synthetic chemicals used in industry that may pose a genetic hazard in our environment is subject of great concern at present. In this respect, the genetic toxicity of fenpropathrin ((RS)-.alpha.-cyano-3-phenoxybenzyl-2,2,3,3-tetramethyl cyclopropane carboxylate, CAS No.:39514-41-8), a pyrethroid insecticide, was evaluated in bacterial gene mutation system, chromosome aberration in mammalian cell system and in vivo micronucleus assay with rodents. In bacterial gene mutation assay, no mutagenicity of fenpropathrin (62-$5000\mug/plate$) was observed in Salmonella typhimurium TA 98, 100, 1535 and 1537 both in the absence and in the presence of S-9 metabolic activaton system. In mammalian cell system using chinese hamster lung fibroblast, no clastogenicity of fenpropathrin was also observed both in the absence and in the presence of metabolic activation system in the concentration range of $7-28\mug/ml$. And also, in vivo micronucleus assay using mouse bone marrow cells, fenpropathrin also revealed no mutagenic potential in the dose range of 27-105 mg/kg body weight of fenpropathrin (i.p.). Consequently, no mutagenic potential of fenpropathrin was observed in vitro bacterial, mammalian mutagenicity systems and in vivo micronucleus assay in the dose ranges used in this experiment.

• Imaging Science in Dentistry
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• 제43권3호
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• pp.171-177
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• 2013

Purpose: The purpose of this study was to compare the efficacy of two imaging modes in a cone beam computed tomography (CBCT) system in detecting root fracture in endodontically-treated teeth with fiber posts or screw posts by selecting two fields of view. Materials and Methods: In this study, 78 endodontically-treated single canal premolars were included. A post space was created in all of them. Then the teeth were randomly set in one of 6 artificial dental arches. In 39 of the 78 teeth set in the 6 dental arches, a root fracture was intentionally created. Next, a fiber post and a screw post were cemented into 26 teeth having equal the root fractures. High resolution (HiRes) and standard zoom images were provided by a CBCT device. Upon considering the reconstructed images, two observers in agreement with each other confirmed the presence or absence of root fracture. A McNemar test was used for comparing the results of the two modes. Results: The frequency of making a correct diagnosis using the HiRes zoom imaging mode was 71.8% and in standard zoom was 59%. The overall sensitivity and specificity in diagnosing root fracture in the HiRes mode were 71.79% and 46.15% and in the standard zoom modes were 58.97% and 33.33%, respectively. Conclusion: There were no significant differences between the diagnostic values of the two imaging modes used in the diagnosis of root fracture or in the presence of root canal restorations. In both modes, the most true-positive results were reported in the post space group.

• Journal of Periodontal and Implant Science
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• 제25권2호
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• pp.253-266
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• 1995

Recently, available interests concerning the biologic significance of the extracellular matrix and proliferating cells associated with periodontal disease has been increased. The distribution or expression of cellular proliferation by PCNA, macrophage detection by ${\alpha}$-l-antichymotrypsin, fibronectin playing a important role in host defence mechanisms indirectly, and transglutaminase that cross linked to fibronectin and stimulate fibrin stabilization were studied in inflammed and healthy gingiva. The excised tissue samples were fixed neutral formalin for 24 hours, embedded with paraffin, sectioned at 4-61lffi in thickness, and immunohistochemically processed by LSAB method. The positive reaction to PCNA was localized in the suprabasal and basal layer of inflammed gingiva and an increasing reactivity was observed than healthy gingiva. ${\alpha}$-I-antichymotrypsin positive cells were localized in the basal layer of inflammed gingiva, and there was no or rare positive cells in healthy gingiva. The positive reaction to fibronectin in inflammed gingiva was more than healthy gingiva,"and shown in the connective tissue subjacent to basement membrane of epithelium and in the periphery of the collagen fiber bundles. The positive cells by transglutaminase in inflammed gingiva were noted in suprabasal, spinous, and keratin layer of epithelium, and slightly increased in the capillaries of connective tissues. But the results of this study demonstrated in vitro reaction. Therefore, the role of PCNA,${\alpha}$-l-antichyrnotrypsin, transglutaminase, fibronectin and coefficient with other growth factor and extracellular matrix were further investigated in vivo.

• Nutrition Research and Practice
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• 제7권4호
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• pp.249-255
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• 2013

In this study, the protective effects of EGCG on L-3,4-dihydroxyphenylalanine (L-DOPA)-induced oxidative cell death in catecholaminergic PC12 cells, the in vitro model of Parkinson's disease, were investigated. Treatment with L-DOPA at concentrations higher than $150{\mu}M$ caused cytotoxicity in PC12 cells, as determined using the 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry detection. The apoptotic ratio was similar in cells treated with $100{\mu}M$ EGCG plus $150{\mu}M$ L-DOPA (5.02%) and the control (0.96%) (P > 0.05), and was lower than that of cells treated with L-DOPA only (32.24%, P < 0.05). The generation level of ROS (% of control) in cells treated with EGCG plus L-DOPA was lower than that in cells treated with L-DOPA only (123.90% vs 272.32%, P < 0.05). The optical density in production of TBARS in cells treated with L-DOPA only was higher than that in the control ($0.27{\pm}0.05$ vs $0.08{\pm}0.04$, P < 0.05), and in cells treated with EGCG only ($0.14{\pm}0.02$, P < 0.05), and EGCG plus L-DOPA ($0.13{\pm}0.02$, P < 0.05). The intracellular level of GSH in cells treated with EGCG plus L-DOPA was higher than that in cells treated with L-DOPA only ($233.25{\pm}16.44$ vs $119.23{\pm}10.25$, P < 0.05). These results suggest that EGCG protects against L-DOPA-induced oxidative apoptosis in PC12 cells, and might be a potent neuroprotective agent.