View : 45 Download: 0

Histamine-Releasing Factor에 의한 lgE-의존성 ROS 생성에 관한 연구

Histamine-Releasing Factor에 의한 lgE-의존성 ROS 생성에 관한 연구
Other Titles
Studies on the IgE-dependent generation of ROS by IgE-dependent Histamine Releasing Factor
Issue Date
대학원 분자생명과학부
Histamine-Releasing FactorlgE-의존성ROS 생성
이화여자대학교 대학원
Histamine-releasing factor (HRF)는 1979년 histamine releasing activity(HRA)에 관한 논문에서 처음 기술되어, 분자량 15,000 - 50,000 Da에 이르는 다양한 성격을 가지는 soluble mediator로 mast cell 및 basophil에서 histamine 등의 mediator를 방출시킬 수 있는 것으로 조사되었다. 이 중 IgE의 존재하에서 활성을 보이는 IgE-dependent histamine-releasing factor(IgE-HRF)는 1995년 growth-related protein의 일종인 TCTP (translationally controlled tumor protein), mouse p21, human p23과 같은 단백질임이 밝혀졌고, 이후 많은 연구가 진행되어 왔지만, 아직 수용체 등의 신호 전달 체계는 밝혀지지 않은 실정이다. 본 실험실에서는 yeast two-hybrid assay를 시행한 결과, (Na,K)-ATPase의 3rd cytoplasmic domain와 rat IgE-HRF가 상호 결합함을 알 수 있었고, confocal microscope로 관찰한 결과 IgE-HRF가 세포 내로 들어감으로써 histamine을 방출한다는 결과를 얻은 바 있다. 본 논문에서는 IgE-HRF에 의해 RBL-2H3 cell에서 histamine이 분비될 때 (Na,K)-ATPase의 저해효과로 인한 ion imbalance가 일어나고, DNA chip analysis 결과 IgE-HRF에 의해 glutathion S-transferase가 강하게 발현된다는 등의 결과를 바탕으로 reactive oxygen species (ROS)가 관여하는지 실험한 결과, RBL-2H3에서 IgE-dependent HRF에 의한 ROS 생성을 확인할 수 있었다. N-acetylcysteine (NAC)과 같은 항산화제 처리에 의해 IgE-HRF의 ROS 생성이 소멸되고 histamine 방출도 억제되는 것으로 보아 IgE-HRF에 의한 histamine release에 ROS가 intracellular second messenger로 작용할 가능성을 확인하였다. 최근 E.coli system에서 IgE-HRF 합성을 유도하는 과정에서 endotoxin (LPS; lipopolysaccharide)이 오염되어 cell에 적용시 signalling에 혼선을 빚는다는 발표가 있어, 이에 대한 확인 작업도 수행하였다. 이어서 최근 ROS의 upstream으로 예상되는 여러 inhibitor를 적용하여 ROS에 의한 fluorescence를 측정한 결과, IgE-HRF에 의한 ROS 생성에 phospholipase A_(2) (PLA_(2))-arachidonic acid (AA)-lipoxygenase (LOX) 또는 cyclooxygenase (COX) cascade가 ROS의 source가 된다는 것을 알아내었다. 본 논문에서 두 번째로 IgE-HRF의 regulatory molecule을 찾아보고자 full lenght rat IgE-HRF를 bait로 하여 human kidney cDNA library로부터 yeast two-hybrid screening을 실시하여, IgE-HRF와 결합하는 molecule을 조사하였고, 마지막으로 mouse system에서도 사용할 수 있는 pRSET/mouse HRF를 subcloning하였다.;Histamine-releasing factor (HRF) having histamine releasing activity (HRA) had been first reported in 1979. HRFs are soluble mediators that can release histamine and other mediators from basophils and mast cells and its activity can be different depending on the type of IgE. MacDonald et al. first identified IgE-dependent HRF (IgE-HRF) in the molecular level in 1995 and revealed that IgE-HRF was an identical molecule with growth-related protein TCTP (translationally controlled tumor protein), murine homolog p21, and human homolog p23. The activity of IgE-HRF is affected by the presence of IgE, although IgE-HRF is thought to bind to a specific receptor other than IgE. Until now, IgE-HRF signalling pathway including its receptor remains unclear in spite of numerous studies. In our laboratory, it was found that IgE-HRF interacts with the third cytoplasmic domain of α subunit of (Na,K)ATPase using yeast two-hybrid system, can cross the plasma membrane using confocal microscope, and that it causes ion imbalance like Na^(+), K^(+), Ca^(+2) using dye measurement. It suggests that IgE-HRF causes histamine degranulation by acting as a cytoplasmic repressor of (Na,K)ATPase. Since DNA chip analysis showed that gene expression of the glutathione S-transferase was increased by IgE-HRF and there were many reports about ROS as a signalling molecule rather than as a by-product of metabolism, ROS was investigated as an intracellular messenger involved in the histamine degranulation process by IgE-HRF. In RBL-2H3 cells, ROS were generated by IgE-HRF in the presence of IgE using H_(2)O_(2)-sensitive fluorescence of H_2DCFDA. These effects were blocked by anti-oxidant NAC. NAC also inhibited the histamine release by IgE-HRF in the presence of IgE, but not in the absence of IgE. These results showed that IgE-dependent ROS generation could play a role as an intracellular messenger in histamine release by IgE-HRF. Among many signalling pathways, phospholipase A_(2) (PLA_(2)) -arachidonic acid (AA)-lipoxygenase/ cyclooxygenase (LOX/COX) pathway was thought to be the source of ROS. Firstly, AA-cascade-related inhibitors were tested to see if they could block the generation of ROS by exogenous AA in RBL-2H3 cells. PLA_(2) inhibitor AACOCF_(3), LOX/COX dual inhibitor NDGA, COX inhibitor indomethacin, NS-398, naproxen, and LOX inhibitor AA861 were found to inhibit IgE-HRF-induced ROS. These finding indicates that lipoxygenase and cyclooxygenase from PLA_(2) cascade are the source of IgE-HRF-induced ROS. Secondly, to investigate the molecules that regulate the action of IgE-HRF, yeast two-hybrid screening with IgE-HRF as a bait perforned using human kidney cDNA library. The data from yeast two-hybrid screening shows that IgE-HRF interacts mainly with IgE-HRF by itself, glycolytic enzyme TPI (triosephosphate isomerase), and translation elongation factor, Thirdly, to introduce murine HRF system, pRSET/mouse HRF was subcloned and the mouse HRF proteins were successfully expressed.
Show the fulltext
Appears in Collections:
일반대학원 > 생명·약학부 > Theses_Master
Files in This Item:
There are no files associated with this item.
RIS (EndNote)
XLS (Excel)


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.