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Ultra Real-Time PCR을 활용한 Avian Influenza Virus Subtype의 조기진단법
김상태,김영균,김장수,Kim, Sang-Tae,Kim, Young-Kyoon,Kim, Jang-Su 한국미생물학회 2011 미생물학회지 Vol.47 No.1
조류 인플루엔자 바이러스(AIV) 아형을 ultra-time PCR법(UPCR)을 이용하여 초스피드로 진단할 수 있는 방법을 고안하였다. 표적 대상의 프라이머는 AIV H5N1 아형의 hemagglutinin(HA) 유전자 중 가장 상보성이 높은 133 bp의 부위를 선택하였고, 실험의 안전을 위하여 인공합성의 방법으로 제작하였다. 압타머와 결합한 molecular beacon 기반 Mini-Opticon Q-PCR 기기를 사용한 UPCR법으로, 총 UPCR 반응액의 양을 10 ${\mu}l$으로, UPCR과 용융온도 분석시간을 15분 이내로 매우 짧게 단축시켰다. 민감도 측정에서 최소의 주형인 5분자의 HA 유전자만으로 정확히 AIV의 특이적 133 bp를 합성하였다. UPCR로 디자인된 이 PCR은 AIV 아형의 진단에 적용될 수 있을 뿐 아니라, UPCR이 기반되는 진단을 이용하여 다른 병원체에도 널리 적용 될 수 있을 것으로 기대된다. This ultra real-time PCR (UPCR) based diagnosis system for avian influenza A virus (AIV) subtype was designed. The target primer in this study was derived from H5N1 subtype-specific 133 bp partial gene of hemagglutinin (HA), and was synthesized by using PCR-based gene synthesis on the ground of safety. UPCR was operated by Mini-Opticon Q-PCR Quantitative Thermal Cycler using aptamer-based molecular beacon, total 10 ${\mu}l$ of reaction mixture with extraordinarily short time in each steps in PCR. The detection including UPCR and analysis of melting temperature was totally operated within 15 min. The AIV-specific 133 bp PCR product was correctly amplified until 5 molecules of HA gene as minimum of templates. This kind of PCR was drafted as UPCR in this study and it could be used to detect not only AIV subtype, but also other pathogens using UPCR-based diagnosis.
피로수명예측을 위한 잔류강도 저하모델의 파라미터 결정법 제안(II)
김상태,장성수,Kim, Sang-Tae,Jang, Seong-Su 대한기계학회 2001 大韓機械學會論文集A Vol.25 No.9
A new method of parameter determination in the fatigue residual strength degradation model is proposed. The new method and minimization technique is compared experimentally to account for the effect of tension-compression fatigue loading of spheroidal graphite cast iron and graphite/epoxy laminate. It is shown that the correlation between the experimental results and the theoretical prediction on the fatigue life and residual strength distribution using the proposed method is very reasonable. Therefore, the proposed method is more adjustable in the determination of the parameter than minimization technique for the prediction of the fatigue characteristics.
김상태,고우찬,이승우,김흥래,Kim, Sang-Tae,Koh, Woo-Chan,Lee, Seung-Woo,Kim, Heung-Rae 한국지하수토양환경학회 2015 지하수토양환경 Vol.20 No.7
In this study, we have developed the performance evaluation model for the optimal soil remediation technology selection. Performance evaluation model is composed in the evaluation of two steps. In the first stage, the candidate technologies are derived according to the conditions of drilling, type and concentration of pollutants, and the saturated/unsaturated of target site. In the second stage, each individual candidate technology is evaluated by performance evaluation model. The performance evaluation model has 5 groups of evaluation items and 12 evaluation items which have their own evaluation index and their own weights through the AHP approach surveying 40 experts. From the case study of actual design cases, the applicability of the performance evaluation model was confirmed.
김상태,서기석,채영수,엄상철 ( Sang Tae Kim,Kee Suck Suh,Young Soo Chae,Sang Cheul Eom ) 대한피부과학회 1994 대한피부과학회지 Vol.32 No.6
멜라닌 색소의 합성을 조절하는 정확한 기전은 확실치 않지만 유전적 요인 이외에 자외선이나 호르몬과 같은 여러 물리적, 화학적, 생리적 요인이 멜라닌 합성을 조절한다고 알려져 있다. 자외선 조사 후 멜라닌 색소의 증가는 주로 색소세포내 tyrosinase의 활성화와 활동성 멜라닌 세포 수의 증가에 기인한다. 한편 멜라닌 세포의 증식을 감소시키고 멜라닌 합성을 억제하는 화합물로는 hydroquinone, monobenzylether of hydroquinone과 같은 catechol과 phenol화합물이 알려져 있으며 vitamin A 유도체인 trans-retinoic acid와 포화 dicarboxylic acid인 azelaic acid 등도 멜라닌 세포의 증식과 멜라닌 합성을 억제한다고 보고된 바 있다. 최근 arbution(hydroquinone-β-D-glucopyranoside), glycolic acid(GA), kojic acid(KA) 및 pentadecenoic acid(PDA) 등의 색소형성 억제 작용이 제시되고 있다. 이 중 hydroquinone 유도체인 arbutin은 세포 독성이 약하나 색소 형성을 상당히 억제하여 악성 흑생종 세포에서 멜라닌 색소 형성을 저하시킨다는 것이 알려졌으며, 탈피술(chemical peeling)에 사용되고 있는 GA는 eumelanin 합성을 억제하는 vitamin C와 화학적 구조가 유사하므로 GA가 멜라닌 세포와 피부 색조에 영향을 미칠수 있을 것으로 생각되나 확실히 규명된 바 없다. KA[5-hydroxy-2(hydroxymethyl)-4-pyrone]는 구리 이온과 결합하여 tyrosinase의 활성도를 억제하고 dihydroxy indol caboxylic acid로의 전환을 억제함으로서 색소 형성을 저하시킨다고 하며, PDA는 다중부포화 지방산으로 premelanosome의 구조를 변화시키고 멜라닌 색소 중합체의 형성과 peroxidase 활성도를 억제하는 등의 기전으로 색소 형성을 저하시킨다고 추정되고 있다. Arbutine, KA 및 PDA가 흑색종 세포 배양에서 멜라닌 색소 형성을 저하시킨다는 것이 알려졌으나 이들 약물이 배양된 정상 인체 멜라닌 세포에 미치는 영향과 in vivo에서의 멜라닌 세포 수와 형태학적 변화에 미치는 영향은 아직 확실치 않다. 이에 저자들은 배양된 정상 인체 멜라닌 세포와 B-16 흑색종 세포 및 C57EL 흑색 생쥐 피부에 이들 약물을 투여하거나 도포하여 배양된 멜라닌 세포와 B-16 흑색종 세포에서는 세포 수와 멜라닌 양을 측정하고 C57BL 흑색 생쥐에서는 DOPA 염색을 시행하여 멜라닌 세포의 수적, 형태학적 변화를 관찰함으로서 UVB를 조사하지 않은 상태에서의 색소형성 억제 효과를 알아 보고자 하였다. 그리고 배양된 멜라닌 세포와 B-16 흑색종 세포 C57BL 흑색 생쥐 피부 및 인체 피부에 각각 UVB를 조사한 후 이들 약물을 투여하여 UVB 조사에 의한 색소형성에 미치는 영향을 알아보기 위해 본 연구를 시행하였다. Background:Melanin pigmentation palys a major role in normal skin color. The rates of melanin synthesis by melanocytes appear to be regulated by ultraviolet-B UVB) rediation and chemicals, though the precise mechanisms modulating human epidermal pigmentation are unknown. Several chemicals including arbutin, kojic acid(KA), pentadecen?ic acid(PDA) and glycolic acid(GA) have been suggested as a melanogenesis inhibitory compounds because of their chemical or biological similarities with hydroquinone or their tyrosinase inhibitory effcet. Objective:The purpose of this study was to evaluate the inhibitory effect of arbutin, GA, KA and PDA on UV-induced melanogenesis in the in vitro and in vivo pigmentary system. Methods:Cultured normal melanocytes and B-16 melanoma cells, and C57BL mice and human volunteers were used for in vitro and in vivo studies respectively. They were administered to UVB irradiated or nonirradiated cultured normal human melanocytes, and B-16 melanoma cells. For the in vivo study, these chemicals were topically applied to C57BL mice and human ?olunteer skin after UVB irradiation. Numeric and morphologic changes and melanin content were measured in cultured normal human melanocytes and B-16 melanoma cells. In the C57BL mice, numeric and morphologic changes of split-DOPA stained melanocytes were assessed. In the human volunt?rs, gross pigementary changes wre evaluated. Results 1. The number and melanin content of cultured melanocytes initially decreased after UVB-irradiation, but the melanin content increased 5 days after irradiation. 2. Cell numbers of irradiated or nonirradiated cultured human melanocytes decreased in arbutin(10^-3M), KA(10^-3M, 10^-5M), PDA(10^-3M) groups. Those of the cultured B-16 mealnoma cells decreased only in the arbutin(10^-3M) group after UVB irradiation. 3. Melanin contents of cultured human melanocytes decreased in crbutin(10^-3M, 10^-5M), KA(10^-3M, 10^-5M) and PDA(10^-3M) groups. Those of cultured B-16 melanon a cells decreased in arbutin(10^-3M, 10^-5M) groups after UVB-irradiation or nonirradiation. 4. The number of split-DOPA(+) melanocytes decreased in the groups ?reated with KA 1% for 3,5 and 7 weeks, KA 0.1%, arbutin 3%, arbutin 5% for 5and 7 qwwks and PDA 5.0% for 7 weeks in the C57BL mice. 5. The number of split-DOPA(+) melanocytes decreased in the groups ?reated with KA 1.0%, PDA 5.0%, arbutin 3% and arbutin 5% for 5 and 7 weeks and KA 0.1% for 7 weeks in UVB irradiated C57BL mice. 6. Visible inhibition of UVB-induced hyperpigmentation was observed in arbutin applied sites in 4 of the 6 volunteers 3 weeks after the application. GA did not show an inhibitory effect on UVB-induced hyperpigmentation in all subjects. Conclusion:Arbutin, KA, PDA had a suppressive effect on ? of nonirradiated melanocytes and melanoma cells as well as UVB-induced hyperpigmentation. It is suggested that these drugs might be candidates as compounds that may control hyperpigmentary disorders.(Kor J Dermatol 1994;32(6):977~989)
자외선 B조사 후의 인체 각질형성세포 배양상청액이 멜라닌세포의 배양에 미치는 영향
김상태,서기석,채영수,조무연,정인철 ( Sang Tae Kim,Kee Suck Suh,Young Soo Chae,Moo Youn Jo,In Cheol Cheong ) 대한피부과학회 1994 대한피부과학회지 Vol.32 No.5
최근 멜라닌세포와 각질형성세포간의 밀접한 관계로 미루어 각질형성세포가 멜라닌세포의 증식과 기능을 조정할 것이라고 일부 추정되고 있다. 즉 각질형성세포가 정상 상태나 염증 상태에서 여러 cytokine을 생산하는 것으로 알려져 있는데, 이중 interleukin(IL)-1 및 IL-6 등이 멜라닌세포의 증식과 기능에 영향을 미친다는 보고가 있다. DeLuca 등은 세포들 사이의 직접적인 접촉이 멜라닌세포의 성장 및 분화에 필수적이라고 주장하였으며 Gordon 등은 각질형성세포의 배양상청액을 멜라닌세표 배양액에 첨가시 멜라닌세포의 증식과 멜라닌(melanin) 합성을 자극한다고 보고한 바 있다. 자외선 조사에 의한 피부 색소침착 반응 중 ultra-violet-B(UVB)에 의한 지연 색소침착은 멜라닌세포의 수와 멜라닌 합성이 증가되고, 멜라닌세포에서 각질형성세포로 멜라닌 색소의 이동이 증가되는 등의 과정에 의해 발생하게 되며, 이러한 색소침착은 인체에 유해한 작용을 일으키는 과도한 자외선을 흡수하고 차단하는 광보호 작용을 나타내게 된다. 그러나 UVB 조사에 의한 멜라닌 합성의 증가 및 각질형성 세포로 멜라닌의 이동을 증가시키는 기전과 조절인자는 아직 확실히 알려져 있지 않다. 현재까지 UVB가 일차적으로 멜라닌세포에 작용하여 멜라닌세포의 수와 멜라닌 합성이 증가됨으로서 색소침착이 야기된다고 생각되어 왔으나, 표피를 구성하는 세포의 대부분이 각질형성세포이고 또 피부색소 형성에 효과적인 UVB는 표피하부에 위치한 멜라닌세포보다는 주로 각질형성세포가 접하게 되므로 자외선 조사시의 색소형성 과정에 각질형성세포가 일차적 역할을 담당할 가능성이 있을 것으로 저자들은 생각하고, UVB를 조사하거나 조사하지 않은 각질형성세포의 배양상청액을 멜라닌세포 배양에 투여한 후 멜라닌세포의 성장과 멜라닌 합성에 미치는 영향을 비교, 관찰함으로서 정상 피부 색조계에 있어 각질형성세포와 멜라닌세포와의 기능적 관계를 규명하고, UVB에 의한 피부 색소 형성에 각질형성세포가 미치는 영향을 알아보고자 본 연구를 시행하였다. Background:Melanin pigment palys a major role in the expression of normal human skin color as well as in the photoprotection against ultraviolet damage. Melanin produced in melanocytes is transferred via dendrites to surrounding keratinocytes, and this anatomical relationship is termed as epidermal melanin unit. The rates of pigment synthesis and transfer by me anocytes appear to be influenced by ultraviolet light, though the precise factors regulating human epidermal pigmentation remain unelucidated. It has been reported that keratinocytes in vitro release factors that could modulate melanocyte behavior. Ultraviloet irradiation was also been known to enhance the release of various kinds of cytokine from keratinocytes in vivo and in vitro. Objective:We postulated that keratinocytes rather than melanocytes could play a primary role in UVB-induced pigmentation and keratinocytes, when irradiated with UVB, release substances that could modulate or stimulate melanin synthesis from melanocytes. The fact that keratinocytes are located efficiently for direct sunlight irradiation at the top of melanocytes that they release various biological factors known to stimulate melanin synthesis from melanocytes and that they constitute the majority of epidermal cells supported this possibility. To investigate this possibility, we evaluated the effect of supernatant from UVB-irradiated cultured keratinocytes on the growth, melanin content, and tyrosinase activity of human melanocytes. Methods:Human cultured keratinocytes were irradiated with UVB 30, 60, or 120mJ/㎠) once, and after 24 hours, supernatant of the keratinocytes were collceted and added to a growth medium of melanocytes for 5 days in concentration of 15, 25 or 35%, We observed numeric and morphologic changes as well as melanin content and tyrosinase activity in situ of cultured human melanocytes. Results: 1. When cultured melanocytes were incubated with supernatant of non-irradiated keratinocytes, the number of melanocytes, amount of melanin and tyrosinase activity increased in groups added with 25% or 35% concentration of supernatant. 2. The number of melanocytes incubated with 15% or 25% concen rations of supernatant from cultured kiratinocytes irradiated with UVB increased in both 30 and 60mJ/㎠ of UVB irradiated groups and decreased in 120mJ/㎠ of UVB irradiated groups. 3. The melanin content of melanocytes incubated with 15% concentration supernatant from UVB-irradiated cultured keratimocytes increased in 120mJ/㎠ of UVB irradiated groups and the melanin content of melanocytes incubated with 25% concentration of supernatant from UVB-irradiated cultured keratinocytes increased in 60 and 120mJ/㎠ of UVB irradiated groups. When cultured melanocytes were incubated with 35% supernatant concentration of supernatant from UVB-irrdiated keratinocytes, the melanin content increased in 30mJ/㎠ of UVB irradiated groups. 4. The tyrosinasse activity of melanocytes incubated with 15% concentration of supernatant from UVB-irradiated cultured kerainocytes increased in 120mJ/㎠ of UVB irradiated groups and the tyrosinase activity of melanocytes incubated with 25% concentration of supernatant from UVB-irradiated cultured keratinocytes increased in 60 and 120mJ/㎠ of UVB irradiated groups. When cultured melanocytes were incubated with 35% supernatant concentration of supernatant from UVB-irradiated keratinocytes, the tyrosinase activity increased in 30mJ/㎠ of UVB irradiated groups. Conclusion:The above results suggest that UVB-irradiated keratinocytes release soluble or photoactivated factors which could modulate the growth and melanization of melanocytes, and that keratinocytes play an important or primary role in the regulation of UVB induced pigmentation. (Kor J Dermatol 1994;32(5):809~819)