폐상피세포에서 Paraquat에 의한 아포프토시스에 관한 연구
Paraquat-Induced Apoptotic Cell Death in Lung Epithelial Cells
박재석 (단국대학교)
지영구 (단국대학교)
이계영 (건국대학교)
양주연 (단국의대)
정인국 (단국의대)
김윤섭 (단국의대)
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초록
연구배경 : Paraquat는 P-450 reductase에 의해 반응성 산소유리기(ROS)를 발생시켜 세포막, 단백질, 핵산 등과 반응함으로써 세포손상을 유도하며 급성 폐 손상을 일으킨다. 최근 급성 폐 손상 및 급성 호흡곤란 증후군에 있어서 폐상피세포의 아포프토시스가 중요한 역할을 한다고 알려지기 시작하였다. 이에 반응성 산소유리기에 의한 폐 손상의 대표적 물질인 paraquat로 인한 폐상피세포의 세포죽음이 아포프토시스인지 확인하고 dexamethasone, N-acetylcysteine, 그리고 bcl-2가 paraquat로 인한 폐상피 세포죽음에 어떠한 영향을 미치는지 등을 연구하였다.방법 : 폐상피세포주인 A549와 BEAS-2B 세포주, 그리고 bcl-2 construct를 유전자 주입한 A549 pcDNA3-bcl-2 세포주를 이용하였다. 아포프토시스는 Annexin Ⅴ assay를 이용하여서 판정하였으며 세포독성 검사는 MTT assay를 이용하였다. Paraquat는 0, 1 μM, 10 μM, 100 μM, 1 mM, 10 mM의 농도로 사용하였다. Dexamethasone은 1 μM의 농도로 paraquat 투여 12시간 전에 전처치하였고, N-acetylcysteine은 1 mM의 농도로 paraquat 투여 1시간 전에 전처치하였다.결과 : 양 세포주 모두에서 paraquat는 농도와 시간 경과에 따라서 세포죽음을 증가시켰고, 이러한 세포죽음은 아포프토시스였다. N-acetylcysteine과 dexamethasone은 시간과 농도에 따라 약간의 차이가 있으나 전반적으로 10~30%의 방어효과가 있었다. Bcl-2를 과발현시킨 A549-bcl-2 세포주에서 A549-neo 세포주에 비해 paraquat에 의한 세포독성이 약 20~30% 정도 차단되었다.
- keywords
- Paraquat, Apoptosis, Bcl-2, N-acetylcysteine, Dexamethasone., Paraquat, Apoptosis, Bcl-2, N-acetylcysteine, Dexamethasone.
Abstract
Background: Paraquat is extremely toxic chemical material, which generates reactive oxygen species (ROS), causing multiple organ failure. In particular, paraquat leads to irreversible progressive pulmonary fibrosis. Exaggerated cell deaths exceeding the normal repair of type Ⅱ pneumocytes leads to mesenchymal cells proliferation and fibrosis. This study examined the followings; ⅰ) whether or not paraquat induces cell death in lung epithelial cells; ⅱ) whether or not paraquat-induced cell deaths are apoptosis or necrosis; and ⅲ) the effects of N-acetylcysteine, dexamethasone, and bcl-2 on paraquat-induced cell deaths. Methods: A549 and BEAS-2B lung epithelial cell lines were used. The cell viability and apoptosis were evalluated using a MTT assay, Annexin Ⅴ staining was monitored by fluorescence microscopy, The level of bcl-2 inhibition was examined by establishing stable A549 pcDNA3-bcl-2 cell lines throung the transfection of pcDNA3-bcl-2 with the mock. Results: Paraquat decreased the cell viability in A549 and BEAS-2B cells in a dose and time dependent manner. The Annexin Ⅴ assay showed that apoptosis was the type of paraquat-induced cell death. Paraquat-induced cell deaths was significantly inhibited by N-acetylcysteine, dexamethasone, and bcl-2 overexpression. The cell viability of A549 cells treated with N-acetylcysteine, and dexamethasone on the paraquat-induced cell deaths were increased significantly by 10 ~ 20%, particularly at high doses. In addition, the cell viability of A549 pcDNA3-bcl-2 cells overexpressing bcl-2 was significantly higher than the untransfected A549 cells. Conclusion: Paraquat induces apoptotic cell deaths in lung epithelial cells in a dose and time dependent manner. The paraquat-induced apoptosis of lung epithelial cells might occur through the mitochondrial pathway. (Tuberc Respir Dis 2006; 61: 366-373)
- keywords
- Paraquat, Apoptosis, Bcl-2, N-acetylcysteine, Dexamethasone., Paraquat, Apoptosis, Bcl-2, N-acetylcysteine, Dexamethasone.
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