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세포기반분석을 이용한 흰쥐신경교종세포에서의 실시간 카스파제-3 활성 측정

Title
세포기반분석을 이용한 흰쥐신경교종세포에서의 실시간 카스파제-3 활성 측정
Other Titles
Real Time Measurement of Caspase-3 Activity Using Cell-Based Assay in Rat Glioma Cells
Author
송영미
Alternative Author(s)
Song, Young-Mi
Advisor(s)
채영규
Issue Date
2007-02
Publisher
한양대학교
Degree
Master
Abstract
세포기반분석 (cell-based assay)은 살아 있는 세포를 사용하여 특정 유전자의 발현이나 단백질의 기능 등을 분석 하는 것이다. 본 실험에서는 살아있는 세포에서 실시간으로 카스파제-3 활성을 측정하기 위하여 pCaspase 발현 벡터를 형질전환시켰다. 형질전환한 세포는 카스파제-3가 활성이 되면 green fluorescent protein (GFP)가 세포질에서 핵으로 이동하는 특징을 가지고 있다. 구축한 세포주에 staurosporine (STP)을 다양한 농도로 처리하여 공초점 현미경으로 카스파제-3 활성을 이미지로 측정 및 분석하였다. 그 결과 STP의 농도와 처리시간이 증가함에 따라 GFP가 세포질에서 핵으로 이동된 세포의 숫자가 증가함을 확인하였다. Propidium iodide (PI)를 동시에 처리한 경우, 죽은 세포는 DNA가 PI에 의해 염색이 되고 세포자멸사에 의해 죽은 세포의 경우는 염색된 PI와 세포질에서 핵으로 이동한 GFP가 함께 관찰되어 노란색으로 보였다. 본 연구에서 개발한 카스파제-3 활성 측정용 세포주는 세포자멸사를 살아 있는 세포에서 실시간으로 스크리닝할 수 있다.
Cell-based assay system is tool to understand intracellular events and protein functions in living cell requires real-time monitoring using the imaging of fluorescence. This study is contents about detection of apoptosis using real time cell-based assay system in living cells. We constructed C-6-caspase sensing cells using caspase-3 sensing system. Caspase-3 assay system was composed of nuclear export signal (NES), DEVD, green fluorescent protein (GFP) and nuclear localization signal (NLS). When caspase-3 is active, the NES is cleaved off and the NLS promotes transport the GFP into the nucleus from the cytosol. C6-caspase-3 sensing cell was treated with staurosporine (STP) to induce apoptosis. Translocation of GFP was screened using confocal microscopy treated with different concentration of STP and propidium iodide (PI). After treatment of STP, the GFP signal in cytosol decreased noticeably, whereas it increased gradually in the nucleus. C6-caspase-3 sensing cell was treated with STP to induce apoptosis and stained PI that indicates the late stage of apoptosis. Translocation of GFP and PI was screened by using confocal microscopy. At 24hr post treatment of STP, most (ca. 80%) of the GFP intensity was detected with PI in nucleus, which meant the apoptosis was completed. Our results suggest that these approaches are a useful tool to study on caspase-3 activity in living cell and may be an important process for the drug screening system in living cell.
URI
https://repository.hanyang.ac.kr/handle/20.500.11754/150119http://hanyang.dcollection.net/common/orgView/200000406094
Appears in Collections:
GRADUATE SCHOOL[S](대학원) > DEPARTMENT OF BIOCHEMISTRY(생화학과) > Theses (Master)
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