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생쥐 수정란의 동결보존시 Electron Microscope (EM) Grid를 이용한 초자희동결법의 효용성에 관한 연구
이은정(Eun-Jeong Lee),문옥성(Og-Sung Moon),최양규(Yang-Kyu Choi),김형진(Hyoung-Chin Kim),송해범(Hai-Bom Song),박창식(Chang-Sik Park) 한국실험동물학회 2004 Laboratory Animal Research Vol.20 No.2
본 연구는 EM grid를 이용한 생쥐 수정란의 초자화동결을 통해 수정란 동결보존에 대한 효율적인 방법을 모색하고자 수행되었다. 연구에 사용된 수정란은 총 2,744개이었으며, 동결방법에 따른 동결-융해 후 생존율과 72시간 동안 배양한 후 체외 발달율을 비교 검토하였다. 초자화동결보존액과 융해액에 노출만 시킨 수정란의 생존율은 plastic straw를 시용한 초자화동결 Ⅰ(VM Ⅰ)이 90.4%이고 EM grid를 사용한 초자회동결 Ⅱ(VM Ⅱ)가 93.3%로 두 군 간에는 차이가 없었고, 체외 발달율(각각 76.4%와 78.0%)에서도 차이가 나타나지 않았다. 동결-융해과정을 거친 후 수정란의 생존율은 완만동결에서 81.8%, 에서 76.5% 그리고 VM Ⅱ에서 82.7%로서 유의한 차이를 보이지 않았다. 체외 배양 72시간째 배반포기 배로의 발달에서는 VM Ⅱ(72.6%)가 완만동결(65.1 %)과 VM Ⅰ(62.5%)에 비해 높은 발달율을 보였다. 실온에서 EM grid를 이용하여 초자화동결을 할 때 EG20에서 3분, EFS40에서 0.5분 동안 노출시킨 실험군이 가장 높은 생존율(83.1%)과 체외 발달율(74.1%)을 나타내었고, 37℃ 조건에서는 EG20에 2분, EFS40에서 0.5분 동안 노출된 실험군이 가장 높은 생존율과 발달율을 보였다. 수정란에 대한 융해액 노출시간에 따른 생존율과 체외 발달율은 3단계의 융해액에서 1.5분씩 노출시킨 실험군에서 가장 높은 생존율(84.3%)을 나타내었고, 0.5분씩 노출시킨 실험군은 가장 낮은 생존율(73.8%)을 나타내었다. 체외 배양 72시간째에 배반포기 배로의 발달율에서 단계별 융해액에 2분씩 노출시킨 실험군에서 가장 높은 발달율을 나타내었지만 그룹간에 유의한 차이는 없었으며, 단계별 융해액에 0.5분씩 노출시킨 실험군이 역시 가장 낮은 발달율을 나타내었다. This present study was conducted to examine the effectiveness of vitrification by electron microscope (EM) grid in cryopreservation of mouse 2cell embryos. A total of 2,744 mouse 2-cell embryos were used, and the rate of survival and development in vitro after freezing-thawing by three different cryopreservation methods were comparatively examined. In the experiment of only exposure on freezing and thawing solution, the survival rates of 2-cell mice embryos were not significant difference between vitrification method (VM) Ⅰ and Ⅱ (90.4% and 93.3%, respectively), and also, the rates of development into blastocyst were not significant difference between two groups (76.4% and 78.0%, respectively) after 72h in culture in vitro. The survival rates of frozen-thawed embryos between slow-freezing (81.8%), VM Ⅰ (76.5%) and Ⅱ (82.7%) were not significances. In the developmental rate into blastocyst, VM Ⅱ was highest between three experimental groups, but there were no significances. Using VM Ⅱ at room temperature, in the rates of survival and development into blastocyst by exposure time of embryos on freezing solutions, the embryos exposed for 3 min in EG20 and then 0.5 min in EFS40 were revealed highest (83.1 % and 74.1 %, respectively) except for control. However, at 37℃, in the rates of survival and development into blastocyst, the embryos exposed for 2 min in EG20 and then 0.5 min in EFS40 was revealed highest (80.2% and 74.1%, respectively) except for control. In the survival rate by exposure time of embryos on thawing solutions, the embryos exposed sequentially intervals 1.5 min was highest (84.3%), however, in the developmental rate into blastocyst, the embryos exposed sequentially intervals 2 min was highest (74.6%).
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연구논문 : 마우스 배아줄기세포를 이용한 심근세포 분화촉진 물질 탐색
남기환 ( Ki Hoan Nam ),김은경 ( Eun Kyoung Kim ),원영석 ( Young Suk Won ),문옥성 ( Ok Seong Moon ),안병우 ( Byeong Woo Ahn ),김형진 ( Hyoung Chin Kim ) 한국동물실험대체법학회 2007 동물실험대체법학회지 Vol.1 No.2
Embryonic stem cells are derived from inner cell mass of blastcyst. They are pluripotent in differentiation and show self-renewal ability. Therefore, the cells can differentiate into cell types which can be derived from all the three germ layers. In this report, we screened 100 compounds for their potential effects on the differentiation of KH2 mouse embryonic stem cells to cardiomyocytes. KH2 cells are developed in our lab and express enhanced green fluorescence protein under the direction of α-myosin heavy chain gene promoter. This cell line shows no fluorescence in undifferentiated condition. However, cardiomyocytes derived from the cells show green fluorescence. Test compound was added during the differentiation period of the cells. After 15 days of differentiation, the cells were trypsinized and measured by flow cytometry for their fluorescence protein expression. All tested compound did not show any significant increase in the rate of fluorescent cells compared with that of their control group. However, we could confirm that our cell screening system is very stable.
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마우스 배아줄기세포를 이용한 심근세포 분화촉진 물질 탐색
남기환 ( Ki Hoan Nam ),김은경 ( Eun Kyoung Kim ),원영석 ( Young Suk Won ),문옥성 ( Ok Seong Moon ),안병우 ( Byeong Woo Ahn ),김형진 ( Hyoung Chin Kim ) 한국동물실험대체법학회 2007 동물실험대체법학회지 Vol.1 No.2
Embryonic stem cells are derived from inner cell mass of blastcyst. They are pluripotent in differentiation and show self-renewal ability. Therefore, the cells can differentiate into cell types which can be derived from all the three germ layers. In this report, we screened 100 compounds for their potential effects on the differentiation of KH2 mouse embryonic stem cells to cardiomyocytes. KH2 cells are developed in our lab and express enhanced green fluorescence protein under the direction of α-myosin heavy chain gene promoter. This cell line shows no fluorescence in undifferentiated condition. However, cardiomyocytes derived from the cells show green fluorescence. Test compound was added during the differentiation period of the cells. After 15 days of differentiation, the cells were trypsinized and measured by flow cytometry for their fluorescence protein expression. All tested compound did not show any significant increase in the rate of fluorescent cells compared with that of their control group. However, we could confirm that our cell screening system is very stable.
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마우스배아줄기세포의 in vitro 시험계 활용을 위한 신경세포 분화프로토콜의 비교
김해림 ( Hae Rim Kim ),남기환 ( Ki Hoan Nam ),김은경 ( Eun Kyoung Kim ),윤원기 ( Won Ki Yoon ),원영석 ( Young Suk Won ),문옥성 ( Ok Seong Moon ),정의배 ( Eu Bae Joung ),안병우 ( Byeong Woo Ahn ),김형진 ( Hyoung Chin Kim ) 한국동물실험대체법학회 2008 동물실험대체법학회지 Vol.2 No.1
Mouse embryonic stem cells are pluripotent stem cells that can be differentiate into all the cell types derived from three germ layers in vitro. We aimed to confirm the neuronal cell types derived from the embryonic stem cells by two different differentiation protocols, which would guide us which protocol is useful for in vitro neuronal toxicity test. The mouse embryonic stem cells derived from 129 mouse strain, TC-1 cells, were differentiated according to 30-day differentiation protocol or 15-day differentiation protocol. At the end of the differentiation period, neuronal cells (neuron, astrocyte and oligodendrocyte) were identified by immunocytochemistry using marker antibodies. According to the results, the numbers of astrocytes and oligodendrocytes were much higher than that of neurons in 30-day differntiation protocol. However, oligodendrocytes were overwhelming compared to astrocytes and neurons in the 15-day differntiation protocol. These results indicated that the neuronal cell types and the cell numbers derived from the embryonic stem cells should be considered when selecting in vitro neuronal cell toxicity models using embryonic stem.
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