천연물이 장상피세포의 염증과 장 투과성에 미치는 영향 : Effect of Natural Products on the Inflammation and Permeability of Intestinal Epithelial Cells
- 주제(키워드) 염증성장질환 , 천연물 , 장투과성 , tight junction
- 발행기관 강릉원주대학교
- 지도교수 강경수,김정호
- 발행년도 2018
- 학위수여년월 2018. 2
- 학위명 석사
- 학과 및 전공 일반대학원 KIST강릉분원학.연협동과정
- 세부전공 해양생물공학전공
- 본문언어 한국어
- 저작권 강릉원주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
염증성 장질환(inflammatory bowel disease, IBD)은 장내미생물과 장상피세포와의 정상적인 상호작용이 무너져 장상피세포에서 염증반응이 과도하게 유발되거나, 장상피세포에서 장 투과성(intestinal permeability)이 비정상적으로 증가하여 이러한 과도한 염증반응이 더욱 악화되어 발병하는 것으로 알려져 있다. 본 연구에서는 농식품 유래의 천연물을 대상으로 이러한 염증성 장 질환의 예방 및 치료 효능을 가지는 물질을 발굴하고자 하였다. 이를 검증하기 위한 모델로써, 사람의 장점막으로부터 유래한 HT-29 세포와 Caco-2 세포주를 배양하여 이용하였다. 그리고 장상피세포의 염증을 유발하기 위해 염증성 사이토카인 (pro-inflammatory cytokine)인 Interleukin-1β (IL-1β)을 이용하였는데, IL-1β는 장상피세포의 초기 염증 반응을 유발하고 특히 염증성 장질환에서 염증 반응을 유도하는 주요 인자로 알려져 있기 때문이다. 약 100여종의 천연물을 탐색한 결과, 오미자 유래의 schisandrin C와 십자화과 채소의 소화대사 성분 중 하나인 3,3’-Diinodolylmethane (DIM)의 장상피세포에 대한 항염증 활성을 확인 할 수 있었다. Schisandrin C와 DIM은 HT-29 및 Caco-2 세포에서 IL-1β에 의해 유도된 염증성 사이토카인인 IL-8의 생성을 현저하게 감소시켰다. 또한 schisandrin C와 DIM은 분화된 장상피세포에서 IL-1β 투여에 의해 증가된 장 투과성을 현저히 감소시켰다. 뿐만 아니라 schisandrin C와 DIM이 염증 및 tight junction관련 단백질의 발현양에 미치는 영향을 확인한 결과, 이 두 천연물은 IL-1β에 의해 증가된 cyclooxygenase-2 (COX-2)발현양을 감소시켰으며, tight junction관련 단백질의 발현양을 조절하였다. 요약하면, schisandrin C와 DIM은 장상피세포에서 항염증 효능을 보이며, 염증 반응에 의해 유발된 비정상적인 장 투과성 증가를 회복시키는 효능이 있었다. 따라서 본 연구에서 발굴한 농식품 유래의 schisandrin C와 DIM은 염증성 장질환을 완화하는 후보 물질로 유용하게 이용될 수 있을 것이다.
more목차
Abstract··························································································ⅰ
List of Figures···················································································ⅲ
국문 초록····················································································· · 01
Ⅰ. 서론(Introduction)·········································································· 02
II. 재료 및 방법 (Materials & Methods)·················································06
2.1. 시료····················································································06
2.2. 세포 배양 조건···································································· 06
2.3. 세포 생존율(cell viability)························································07
2.4. Interleukin 8 (IL-8) 생성량 측정··················································08
2.5. 상피전위 전기저항(transepithelial electrical resistance, TEER) 측정·····10
2.6. 장 투과성(permeability) 측정····················································10
2.7. Western blot을 이용한 inflammation과 tight junction, mucin단백질 발현
측정····················································································11
2.8. 통계분석··············································································11
Ⅲ. 결과(Results)··············································································13
3.1. 장상피세포에서 염증 억제 활성 평가 실험 조건 설정················13
3.1.1. Aspirin을 24시간동안 전처리하고 IL-1β을 6 시간 동안 추가
투여한 조건에서 항염증 효과···················································15
3.1.2. IL-1β를 24시간동안 처리하고 aspirin을 24시간 동안 교체
투여한 조건에서 항염증 효과·············································20
3.1.3. 24시간 동안 IL-1β를 전처리 한 다음, aspirin과 IL-1β를 24시간동안 동시 투여한 조건에서의 항염증 효과···············25
3.2. 다양한 천연물을 대상으로 장상피세포에서의 항염증 효과
스크리닝············································································30
3.3. HT-29 세포에서 schisandrin C가 IL-1β에 의해 유도된 염증에 미치는
영향 검증·············································································30
3.4. 3,3’-Diindolylmethane (DIM)의 장상피세포에서 염증 억제효과·············33
3.4.1. HT-29 세포에서 DIM이 IL-1β에 의해 유도된 염증에 미치는 영향 검증················································································33
3.4.2. Caco-2 세포에서 DIM이 IL-1β에 의해 유도된 염증에 미치는 영향 검증··············································································· 33
3.5. 기타 천연물의 장상피세포에서 염증 억제 효과······························38
3.5.1. HT-29 세포에서 gomisin N이 IL-1β에 의해 유도된 염증에 미치는 영향 검증········································································38
3.5.2. HT-29 세포에서 schisandrin A가 IL-1β에 의해 유도된 염증에 미치는 영향 검증·····························································41
3.5.3. Caco-2 세포에서 fisetin 이 IL-1β에 의해 유도된 염증에 미치는 영향 검증········································································44
3.6. 분화된 장상피세포에서 schisandrin C가 장 투과성(intestinal permeability)에 미치는 영향검증················································47
3.6.1. 분화된 장상피세포에서 schisandrin C가 장상피 두께와 연관된 지표인 transepithelial electrical resistance (TEER)값에 미치는 영향 검증················································································47
3.6.2. FITC-dextran 투과율 측정을 통한 schisandrin C가 분화된 장상피세포의 장 투과성에 미치는 영향 검증························50
3.7. 분화된 장상피세포에서 DIM이 장 투과성(intestinal permeability)에 미치는 영향 검증···································································53
3.7.1. 분화된 장상피세포에서 DIM이 장상피 두께와 연관된 지표인 transepithelial electrical resistance (TEER)값 에 미치는 영향 검증···············································································53
3.7.2. FITC-dextran 기법을 통한 DIM이 분화된 장상피세포의 장 투과성에 미치는 영향 검증················································56
3.8. 분화된 장상피세포에서 DIM이 장 투과성(intestinal permeability)에 미치는 영향검증·····································································59
3.8.1. Schisandrin C가 HT-29 세포와 Caco-2 세포의 염증 및 관련 단백질 발현에 미치는 영향 검증···················································59
3.8.2. DIM이 HT-29 세포와 Caco-2 세포의 염증 및 관련 단백질 발현에 미치는 영향 검증······························································62
3.9. 분화된 장상피세포에서 DIM이 장 투과성(intestinal permeability)에 미치는 영향 검증································································ 65
3.9.1. Schisandrin C가 Caco-2 세포의 tight junction 관련 단백질 발현에 미치는 영향 검증 ···························································· 65
3.9.2. DIM이 Caco-2 세포의 tight junction 관련 단백질 발현에 미치는 영향 검증········································································ 68
Ⅳ. 고찰(Discussion)··········································································71
Ⅴ. 결론(Conclusion)········································································· 75
Ⅵ. 참고 문헌(References)··································································77
