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폐섬유모세포에서 발현되는 ADAM33과 TGF-beta의 조절 작용기전
Reports NRF is supported by Research Projects( 폐섬유모세포에서 발현되는 ADAM33과 TGF-beta의 조절 작용기전 | 2005 Year | 박성우(순천향대학교) ) data is submitted to the NRF Project Results
Researcher who has been awarded a research grant by Humanities and Social Studies Support Program of NRF has to submit an end product within 6 months(* depend on the form of business)
사업별 신청요강보기
  • Researchers have entered the information directly to the NRF of Korea research support system
Project Number E00017
Year(selected) 2005 Year
the present condition of Project 종료
State of proposition 재단승인
Completion Date 2006년 08월 21일
Year type 결과보고
Year(final report) 2006년
Research Summary
  • Korean
  • ADAM-33(A Disintegrin and Metalloproteinase) 은 천식의 기도과민성과 관련된 유전자로 알려져 있다. 그러나 최근에 천식에만 국한되지 않는 폐기능과 관련된 유전자로 여러 연구 결과 밝혀졌으며, 이의 평활근, 섬유모세포에서의 선택적인 발현은 천식의 개형 뿐만 아니라 폐섬유화증의 병인에도 관련이 있을 것으로 여겼다.
    폐섬유화증 환자의 일차섬유모세포주 및 MRC-5 cell line에 IL-4, IL-13, IFN-r와 같은 섬유화와 관련된 사이토카인을 처리한 후 ADAM33의 catalytic domain에대해 RT-PCR 결과, alternative splicing form이 나타남을 확인하였다. 이는 IL-4, IL-13처리후 특히 강하게 나타났으며, RPA(Ribonucleotide protection assay)로 비교해본결과 IL-13, IL-4와 같은 profibrotic cytokine에 의해 upregulation이 됨을 확인할 수 있었다. ADAM-33 catalytic domain 에 대한 antibody 를 제작하여 western blot 을 환자의 BALF(기관지폐포 세척액)및 cell lysate에서 시행하여 55Kda부위에서 band를 확인하였고,IL-4, IL-13를 처리한 섬유모세포에서 ADAM 33 단백질 발현이 대조군에비해 증가되어 나타났다. Soluble ADAM-33 단백질은 폐섬유화증에서 강한 발현이 되고 이는 IL-4, IL-13과 같은 섬유화촉진사이토카인에 의해 조절된다.
  • English
  • ADAM33 has been identifiedas a novel asthma susceptibility gene in genome-wide screening and association studies. Recently, ADAM-33 regarded as lung function gene, not asthma specific. High-level expression in smooth muscles and fibroblasts suggest that ADAM33 plays a rolenot only airway remodeling in asthmatics but may involved fibrosis of lung. The ADAM33 protein was identified in the BAL fluids of IPF(idiopathic pulmonary fibrosis) and normal controls by western blotting using antibody against the catalytic and cytoplasmic domain. In this analysis of the BAL fluids from the IPFs using ASP2 antibody, an intense band of approximately 55 kDa appeared. And, this band also appeared in the conditioned medium of primary fibroblast from IPF and MRC-5 cell line. We found that ADAM33 mRNA expression are regulated by cytokines and alternative splicing form were shown using RT-PCR. Most of this alteration occured mainly in catalytic domain. IL-13, IL-4 upregulate whereas IFN-r inhibit alternative splicing in catalytic domain confirmed by RPA(Ribonucleotide protection assay). Using western blotting, we confirmed that ADAM33 protein(catalytic domain) regulated by this cytokines, too. In conclusion, the levels of soluble ADAM33 protein(catalytic domain) and ADAM33 expression are regulated by profibrotic cytokines such as IL-4, IL-13.
Research result report
  • Abstract
  • ADAM33 has been identifiedas a novel asthma susceptibility gene in genome-wide screening and association studies. Recently, ADAM-33 regarded as lung function gene, not asthma specific. High-level expression in smooth muscles and fibroblasts suggest that ADAM33 plays a rolenot only airway remodeling in asthmatics but may involved fibrosis of lung. The ADAM33 protein was identified in the BAL fluids of IPF(idiopathic pulmonary fibrosis) and normal controls by western blotting using antibody against the catalytic and cytoplasmic domain. In this analysis of the BAL fluids from the IPFs using ASP2 antibody, an intense band of approximately 70 kDa appeared. And, this band also appeared in the conditioned medium of primary fibroblast from IPF and MRC-5 cell line. We found that ADAM33 mRNA expression are regulated by cytokines and alternative splicing form were shown using RT-PCR. Most of this alteration occured mainly in catalytic domain. IL-13, IL-4 upregulate whereas IFN-r inhibit alternative splicing in catalytic domain confirmed by RPA(RNase protection assay). Using western blotting, we confirmed that ADAM33 protein(catalytic domain) regulated by this cytokines, too. In conclusion, the levels of soluble ADAM33 protein(catalytic domain) and ADAM33 expression are regulated by profibrotic cytokines such as IL-4, IL-13.
  • Research result and Utilization method
  • 1.연구 결과
    1) Regulation of ADAM33 mRNA expression by cytokines.
    MRC-5 fibroblast를 IL-1, TGF-b, IL-4, IL-13, INF-r 의 사이토카인을 각각 10ng/ml로 처리 후, lysate로 RT-PCR을 시행하였다. 기대와는 달리 TGF-beta는 별다른 변화가없었으나, IL-4, IL-13, IFN-r 처리시 catalytic domain에서 alternative splicing form이 나타났으며,IL-4 로 처리한 경우에 가장 강하게 나타났다.
    2) RPA( Ribonucleotide protection assay)
    alternative splicing form의 양적인 차이를 확인하기위래 RPA를 IL-4, IL-13, IFN-r처리 군에서 시행했으며,
    PCR과 동일한 IL-4, IL-13처리군에서 강한 alternative splicing form을 확인할 수 있었다.
    3)Immunoblot for ADAM33 catalytic domain
    protein expression의 차이를 확인하기위해 catalytic domain에 대한 Ab를 제작하여 BALF와 lysate에서western blot을 시행하였고, IL-13 처리군에서 ADAM 33의 발현이 가장 강하게, IFN-r의 발현은 약함을 알 수 잇었다.
    결과 : IPF human lung fibroblast에서 ADAM33(catalytic domain)의 alternative splicing form이 profibrotic cytokine에 따라 달리 발현됨을 알 수 있었고, 이는 soluble protein(BALF) expression에서도 볼 수 있었다.
    2.활용방안: 이런 ADAM33 catalytic domain의 activity의 차이가 profibrotic cytokine에의해 조절되어 이로인해 폐섬유화를 촉진한다면, ADAM33 catalytic domain에 대한 antibody or blocking peptide를 개발하여 폐섬유화의 치료에 응용할 수 있을것으로 사료된다.

  • Index terms
  • ADAM33, alternative splicing, lung fibrosis, IL-4, IL-13
  • List of digital content of this reports
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