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중합효소연쇄 반응법에 의한 인형거대세포바이러스(human cytomegalovirus)의 신속한 검출
김의종,오명돈,박기호,신형식,이환종,김남중,최강원 대한감염학회 1996 감염 Vol.28 No.4
목 적 : 인형거대세포바이러스(HCMV)는 면역 저하 환자에서 폐렴, 망막염, 간염 등 치명적인 감염증의 원인이다. 전통적인 세포 배양법으로 HCMV를 분리하기까지는 1주에서 4주가 걸리므로 이 바이러스에 의한 질환이 의심되는 경우 신속한 진단법을 이용한 진단이 바람직하다. 저자들은 한국에서 분리되는 야생주 HCMV를 검출하는데 중합효소 연쇄 반응법을 이용할 경우의 진단적 유용성을 알기 위하여, 전통적인 세포배양법으로 HCMV가 분리된 검체를 이용하여 중합효소 연쇄 반응법의 특이도와 민감도를 평가하였다. 방 법 : 국내 야생주 HCMV 15주, 그리고 전통적인 바이러스 배양법으로 HCMV가 분리된 소변 15검체를 대상으로 중합효소 연쇄 반응을 실시하였다. 중합효소 연쇄 반응은 Towne주의 immediate early antigen의 유전자에서 유리된 primer MIE와 AD169주의 immediate early antigen의 유전자에서 유리된 primer IE를 이용하였다. 증폭 산물은 겔 전기영동 후 EtBR으로 염색하여 자외선 하에서 관찰하였다. Primer IE에 의한 증폭산물은 또한, DNA blot hybridization 방법으로 확인하였다. 결 과 : 1) 국내 HCMV 야생주 15주를 대상으로 중합효소 연쇄 반응을 한 결과 primer IE를 이용한 경우에는 100%(15/15)에서 177bp의 증폭 산물이 관찰되었고, 이들 증폭 산물은 모두 probe IE와 보합 결합되었다. primer MIE를 이용한 경우에는 93%(14/15)에서 435bp의 증폭 산물이 관찰되었다. 2) 세포 배양법으로 HCMV가 분리된 소변 15검체를 대상으로 primer IE를 이용하여 중합효소 연쇄 반응을 한 결과 direct gel analysis 법으로는 73%(11/15)에서, 보합 결합법으로는 87%(13/15)에서 관찰되었다. Primer MIE를 이용한 경우에는 direct gel analysis법으로 87%(13/15)에서 435bp의 증폭 산물이 검출되었다. 결 론 : Primer IE와 MIE를 이용한 중합효소 연쇄 반응법은 국내 야생주 HCMV를 신속히 검출하는데 유용한 검사법이다. Background : Human cytomegalovirus(HCMV) can cause pneumonitis, hepatitis, retinitis and other serious diseases in the immunocompromised patients. It takes 1 to 4 weeks to diagnose HCMV infection by conventional virus culture. Therefore, when HCMV diseases are suspected, a rapid diagnostic method such as polymerase chain reaction(PCR), antigen assay or shell vial culture is desirable. We evaluate the sensitivity and specificity of a PCR for the rapid detection of HCMV wild strains in Korea. Methods : We used 2 sets of primers ; primer IE and primer MIE derived from the sequence for immediate early gene of AD169 strain and Towne strain, respectively. Fifteen clinical isolates of HCMV, suspended in MRC-5 cells, were amplified by PCR. Fifteen urine specimens which were positive for HCMV by conventional virus culture were also amplified. Amplification products were analyzed by polyacrylamide gel electrophoresis. The products from PCR with primer IE were also identified by DNA blot hybridization. Results : PCR using primer IE gave the PCR products in all of the 15 HCMV wild strains. All of these were hybridized with probe IE. When primer MIE were used, 93%(14/15) of the wild strains showed amplified bands by direct gel analysis. When the urine specimens were amplified by PCR with primer IE, amplified bands were seen in 73%(11/15) by direct gel analysis ; 87%(13/15) by hybridization method. When primer MIE were used, 87%(13/15) of the urine specimens showed the PCR products by direct gel analysis. Conclusion : Polymerase chain reaction with primer IE and MIE may be a specific and sensitive diagnostic method for rapid detection of HCMV wild strains in Korea.
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.
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.