한국인에서 C-반응 단백 유전자의 전사조절 부위의 다형성과 전신성 홍반성 루푸스 감수성의 연관성
Polymorphism at the Promoter of C-reactive Protein is Associated with Systemic Lupus Erythematosus in Koreans.
- 주제(키워드) 전신성 홍반성 루푸스+C-반응 단백+단일염기다형성
- 발행기관 아주대학교
- 지도교수 서창희
- 발행년도 2006
- 학위수여년월 2006. 2
- 학위명 석사
- 학과 및 전공 일반대학원 의학과
- 본문언어 한국어
초록/요약
목적 : C-반응 단백은 감염이나 염증, 조직 손상 시에 급격하게 증가하는 급성 반응 물질로써 염증성 질환에서 염증 반응의 표지자로 사용되고 있다. 이에 본 연구자는 C-반응 단백의 유전적 다형성이 전신성 홍반성 루푸스의 질병감수성에 기여할 것이라는 가정 하에 C-반응 단백의 전사조절 부위의 단일염기다형성을 확인하고 한국인에서의 빈도를 결정하여 전신성 홍반성 루푸스의 발병기전에서의 C-반응 단백의 역할을 알아보고자 하였다. 재료 및 방법 : 아주대 병원에 내원한 150명의 전신성 홍반성 루푸스 환자와 질병대조군으로 86명의 류마티스 관절염 환자, 그리고 138명의 정상대조군을 대상으로 연구를 수행하였으며, 이들로부터 전혈을 채취한 뒤 genomic DNA를 분리하여 C-반응 단백 유전자의 전사조절부위의 약 1.5kb를 직접염기서열분석을 시행하였다. 확인된 다형성을 대상으로 유전자형을 결정하였으며, 이와 함께 최근 백인에서 보고 된 두 번째 exon부위의 3개의 다형성은 중합연쇄반응-제한절편길이다형성의 방법을 이용하여 유전자형을 결정하였다. 각각의 다형성과 C-반응 단백의 혈청농도를 비교하기 위해 질병 활성이 가장 낮을 때 대상 환자로부터 혈청을 분리하여, 자동화 된 고감도 immunoturbidometric method (Olympus, UK)를 사용하여 측정하였다. 또한 -390 C>A or T 다형성의 유전자형에 따른 전사조절부위의 활성 정도를 비교하기 위해 promoter activity assay시행하였다. 연구결과의 통계학적 분석은 SPSS v11.0 for window의 독립표본 T-Test를 시행하였으며, p값이 0.05 미만인 경우 통계적으로 유의한 것으로 간주하였다. 결과 : C-반응 단백 유전자의 전사조절부위에서 3개의 단일염기다형성을 확인하였고, 이중 -862 T>C, -822 A>G 다형성은 보고 된 바 없는 다형성이었다. CRP의 전사조절부위와 exon에 존재하는 총 6개의 다형성 모두 대립유전자의 빈도 상에서 한국인과 백인 간에 큰 차이를 보였으며, 특히 -822 A>G와 2043A>G의 다형성의 경우 major allele과 minor allele이 바뀌어 나타날 정도로 큰 빈도상 차이를 보였다. 하지만 한국인 전신성 홍반성 루푸스와 정상 대조군 간의 통계적으로 유의한 차이는 오직 -390 C>A or T의 다형성에서만 관찰되었다(re : p=0.025). 이후 전신성 홍반성 루푸스 환자의 혈청 C-반응 단백치와 각각의 다형성의 유전자형과 비교한 결과 -390 C>A or T 다형성과 2043 A>G 다형성의 경우 minor allele 하나 이상 포함한 사람에 있어서 C-반응 단백치가 통계적으로 유의하게 증가되어 있는 것을 확인 할 수 있었다(각각 p=0.023, p=0.020). 또한 루푸스 환자와 정상 대조군 간에도 빈도상 차이를 보였으며, base line C-반응 단백의 발현에도 영향을 미침을 확인한 -390 C>A or T 다형성을 대상으로 promoter activity assay를 시행한 결과 minor allele인 A 또는 T allele을 포함한 promoter reporter construct에서 major allele인 C allele을 포함한 promoter reporter construct보다 promoter activity가 약 3배가량 증가됨을 확인할 수 있었다(p<0.001). 결론 : 따라서 본 연구에서 조사한 C-반응 단백 유전자의 전사조절부위에 존재하는 -390 C>A or T 단일염기 다형성은 C-반응 단백의 발현에도 영향을 미칠 뿐만 아니라, 한국인 전신성 홍반성 루푸스에 대한 질병감수성에 기여할 것으로 사료된다.
more목차
국문요약 ················································································· ⅰ
차 례 ················································································· ⅲ
그림차례 ················································································· ⅴ
표 차례 ················································································· ⅵ
Ⅰ. 서 론 ·················································································· 1
Ⅱ. 재료 및 방법 ········································································· 4
A. 대상환자군의 선별과 genomic DNA 준비 ·································· 4
1. 대상환자군과 대조군 ······················································ 4
2. 환자군과 대조군으로부터 전혈과 혈청 채취 ··························· 4
3. DNA 추출 ·································································· 4
B. 단일염기다형성 탐색 ··························································· 5
1. Primer 디자인과 중합연쇄반응을 통한 증폭 ··························· 5
2. 염기서열분석 ······························································· 5
3. 단일염기다형성 확인 ······················································ 6
C. 단일염기다형성의 유전자형 결정 ············································· 6
1. C-반응 단백 유전자의 전사조절부위에서 확인된 다형성의
유전자형결정 ······························································· 6
2. C-반응 단백체의 전사부위에 존재하는 다형성의 유전자형 결정 ··· 6
D. 단일염기다형성의 유전자형에 따른 혈청 C-반응단백치 ·················· 7
E. -390 C>A or T 다형성에 대한 Promoter reporter assay ··············· 9
1. C-반응 단백의 전사조절부위를 포함한 plasmid 제작 ················ 8
2. Transformation ···························································· 10
3. 집락 선택 ·································································· 10
4. Plasmid DNA 추출 ······················································ 12
5. Transfection ······························································ 12
6. Luciferase assay 와 β-Galactosidase assay ·························· 13
F. 통계분석 ········································································ 13
Ⅲ. 결 과 ················································································ 16
A. C-반응 단백 유전자의 전사조절부위의 단일염기다형성 확인 ·········· 16
B. 단일염기다형성의 유전자형 결정 ··········································· 16
C. C-반응 단백 유전자의 단일염기다형성의 유전자형과 baseline
C-반응 단백치의 상관관계 ·················································· 17
D. -390 C>A or T 다형성에서 각각의 유전자형에 따른
전사조절부위 활성의 비교 ·················································· 17
Ⅳ. 고 찰 ················································································ 24
Ⅴ. 결 론 ················································································ 27
참고문헌 ················································································· 28
ABSTRACT ············································································ 32
