TDI에 의한 직업성 천식에서 TRP channel 의 역할 규명
The role of transient receptor potential in airway epithelial cells in toluene diisocyanate-induced occupational asthma
- 주제(키워드) Airway epithelium , inflammation , toluene 2 , 4-Diisocyanate , occupational asthma , transient receptor potential melastatin family member 8 receptor
- 발행기관 아주대학교
- 지도교수 박해심
- 발행년도 2018
- 학위수여년월 2018. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의학과
- 실제URI http://www.dcollection.net/handler/ajou/000000027477
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Background: Toluene diisocyanate (TDI) is the most important cause of occupational asthma (OA), and various pathogenic mechanisms have been suggested; of which, neurogenic inflammation is an important pathway to induce airway inflammation. Transient Receptor Potential Melastatin 8 (TRPM8) is a well-established cold sensing cation channel and expressed in neuronal cells as well as bronchial epithelial cells. A recent genome-wide association study (GWAS) from TDI-exposed workers showed a significant association for the rs10803666 SNP mapping to the TRPM8 gene. Objective: We hypothesized TRPM8, located in airway epithelial cells may involve in the pathogenic mechanisms of TDI-induced OA and investigated its role. Methods: Bronchial epithelial cells (BEAS-2B) were treated with TDI in dose- and time- dependent manners. The expression levels of mRNA and protein of TRPM8 were determined by quantitative RT-PCR and Western blotting. Its morphological change by TDI was evaluated immunocytochemisty. The alteration of inflammatory cytokines’ transcripts was examined in accordance with TRPM8 activation by TDI. ELISA of TRPM8 was performed using the induced sputum of asthmatics and normal controls. Results: TRPM8 expression at both mRNA and protein levels were noted in airway epithelial cells, which were enhanced by TDI. TRPM8 activation by TDI led to significant increases in mRNA for interleukin (IL)-4, IL-13, IL-25, and IL-33. Increased expressions of the cytokine genes by TDI were partly attenuated after the treatment with a TRPM8 antagonist. In sputum samples, the expression of IL-25, 33, and TSLP mRNA as well as TRPM8 increased in the sputum of asthmatics compared than normal controls. There was a significant association between TRPM8 and IL-25 mRNA expression in asthmatics. Conclusion: TDI exposure induces increased expression of TRPM8 mRNA in airway epithelial cells coupled with the enhanced expression of inflammatory cytokines. In addition, the expression of TRPM8 was increased in the sputum samples in asthmatics as severity increase. These results suggest that TRPM8 may be involved in the epithelial inflammation and subsequent airway remodeling of TDI-OA.
more목차
I. INTRODUCTION 1
II. MATERIALS AND METHODS 5
A. Chemicals 5
B. Cell culture 5
C. Conventional RT-PCR of TRPM8 5
D. Quantitative real-time PCR 6
E. Intracellular reactive oxygen species measurement 7
F. Immunocytochemistry 8
G. Participant selection 9
H. Measurement of cytokine level from sputum specimen 10
I. Statistical analysis 10
J. Ethical issues 11
III. RESULTS 12
A. Identification of TRPM8 transcript and protein in lung epithelial cells 12
B. Effect of TRPM8 transcripts and protein by TDI in lung epithelial cells 14
C. Expression of TRPM8 protein in lung epithelial cells 16
D. Inhibition of TRPM8 transcript and protein by BCTC in lung epithelial cells 18
E. Effect of TRPM8 activation by TDI on the cytokine gene expression in lung epithelial cells 20
F. Measurement of intracellular reactive oxygen species (ROS) production induced by toluene diisocyanate in BEAS-2B cells 25
G. Subject characteristics 27
H. Expression of TRPM8 mRNA and protein from the induced sputum of asthmatic patients 29
I. Increased epithelial driven cytokines in induced sputum from asthmatics and the correlation between TRPM8 and epithelial driven cytokines transcripts 32
IV. DISCUSSION 35
REFERENCES 41
국문요약 49
