병원성 파울러자유아메바에 의해 유도되는 THP-1 표적세포 내 inflammasome 활성
Inflammasome activation in THP-1 target cells triggered by pathogenic Naegleria fowleri
- 주제(키워드) Naegleria fowleri , PAM , caspase-1 , inflammasome , IL-1β , NLRP3
- 발행기관 아주대학교
- 지도교수 신호준
- 발행년도 2015
- 학위수여년월 2015. 8
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000020269
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Inflammasome activation in THP-1 target cells triggered by pathogenic Naegleria fowleri Naegleria fowleri, also known as the brain-eating amoeba, causes acute primary amoebic meningoencephalitis. During swimming and other recreational water activities, N. fowleri trophozoites penetrate the nasal mucosa and invade the olfactory bulbs, resulting in intense inflammatory reactions in the forebrain tissue. To investigate what kinds of inflammasome molecules are expressed in target cells due to N. fowleri infection, human macrophage cells (THP-1 cells) were co-cultured with N. fowleri trophozoites under the non-contact system, and consequently IL-1β production was estimated. By western blotting and ELISA analysis, the caspase-1 activation and IL-1β production from THP-1 cells and the culture supernatant were observed 3 hr after co-cultivation. In addition, the increased expression of ASC and NLRP3, which consist of inflammasome complex, was also observed 3 hr post co-cultivation. To confirm the caspase-1 activation and IL-1β production via NRLP3 inflammasome in THP-1 cells triggered by N. fowleri trophozoites, several inhibitors were pretreated on THP-1 cells. Results of the inhibition assay show that CA-074 (cathepsin B inhibitor), Glybenclamide (NRLP-3 molecule inhibitor) and Z-VAD-FMK (caspase-1 inhibitor) reduced the caspase-1 activation and IL-1β production from THP-1 cells. This study suggests that N. fowleri infection induces the NLRP3-inflammasome complex, which activates the caspase-1 and subsequently produces the IL-1β, thus resulting in inflammation.
more목차
I. INTRODUCTION 1
II. MATERIALS AND METHODS 4
A. Naegleria fowleri culture and lysate preparation 4
B. Culture of macrophage THP-1 cells 4
C. Non-contact culture system 4
D. Cell extraction from THP-1 cells 7
E. SDS-PAGE and Western blotting for detection of inflammasome molecules 7
F. IL-1β cytokine measurement by ELISA 8
G. Measurement of caspase-1 by ELISA 9
H. Inhibition assay 10
I. Statistical analysis 10
III. RESULTS 11
A. IL-1β production in THP-1 cells co-cultured with N. fowleri 11
B. Caspase-1 activation in THP-1 cells co-cultured with N. fowleri 14
C. NLRP3 and ASC expression in THP-1 cells co-cultured with N. fowleri 17
D. Inhibition of IL-1β production in THP-1 cells co-cultured with
N. fowleri 19
E. Inhibition of caspase-1 activation in THP-1 cells co-cultured with
N. fowleri 21
IV. DISCUSSION 23
V. CONCLUSION 28
REFERENCES 29
국문요약 34
