Serum Amyloid A (1-12) 섬유화 평가 및 Serum Amyloid A 섬유질과 금 나노 입자와의 상호작용

국문 초록 (Abstract)

아밀로이드증은 비정상적인 단백질 응집체인 아밀로이드가 각종 장기에 축적되어 염증을 일으키거나 심각한 질병 및 기능장애를 일으키는 증상을 일컫는다. 대표적인 아밀로이드증에는 amyloid-β 의 응집으로 발생되는 알츠하이머 병, α-synuclein 의 응집으로 생기는 파킨슨병 등이 있다. 아밀로이드증은 크게 1차성 아밀로이드증과 2차성 아밀로이드증으로 나뉜다. 알츠하이머나 파킨슨병 같이 비정상적인 면역 글로블린의 생산으로 인한 아밀로이드의 축적을 1차성 아밀로이드증이라 하고, 염증 반응에 의한 염증성 단백질 생성 증가로 인한 아밀로이드 축적을 2차성 아밀로이드증이라고 한다. Serum amyloid A 는 2차성 아밀로이드증의 일종으로, 특히 류마티스 질환과 관련된 아밀로이드로 알려져 있다.
아밀로이드증의 발병이 점점 증가함에 따라 이러한 질병에 대한 연구가 진행되고 있다. 알츠하이머병, 파킨슨병과 달리 연구가 많이 진행 되지 않은 2차성 아밀로이드증 중 특히 Serum Amyloid A fibrils 의 형성 메커니즘에 대한 연구가 요구되는 실정이다. 이에 본 연구에서는 Amyloid fibrils 의 형성 메커니즘 관찰 및 2차적 구조적 변화를 확인하였다. 더 나아가 이러한 Amyloid fibrils 을 간단하고 혁신적인 방법으로 진단 할 수 있는 진단법 개발의 필요성을 충족하기 위하여 금 나노 입자를 이용한 진단 방법을 연구 및 개발하고자 하였다.
금 나노 입자는 단백질의 친수성 부분과 친유성 부분에 모두 잘 붙는 성질을 가지고 있고, 또한 thiol 기가 있는 아미노산을 가지는 단백질 표면에 dative bonding 을 통해 잘 결합 할 수 있다. 이러한 특성을 이용하여 Amyloid fibrils 표면에 금 나노 입자가 잘 결합하여 상호작용 할 것이라 판단하였고, detection tool 로써 활용가치가 있을 것으로 기대되었기에 연구를 진행하였다. 15 nm, 50 nm 지름을 가지는 구형 형태, 그리고 막대모양 형태의 서로 다른 세 가지 형태의 금 나노 입자를 이용하였다. 이 세 가지 다른 종류의 금 나노 입자는 사이즈에 따른 detection 정도의 차이, 모양에 따른 detection 차이를 동시에 확인하기 위해 사용되었다.
Serum Amyloid A fibrils 형성 메커니즘과 단백질의 2차 구조 확인은 AFM, CD Spectroscopy, ThT Assay 를 통해 분석하였다. 형성된 Amyloid fibrils 과 세 가지 다른 종류의 금 나노 입자의 결합 여부는 TEM 으로 확인하였고, UV-Visble spectroscopy 를 이용하여 Amyloid fibrils 에 결합하여 상호작용한 금 나노 입자에 생긴 변화를 확인 하였다. 단백질이 금 나노 입자와 상호작용을 하면 입자 표면의 고유한 전자 분포에 변화가 생기는데, 이러한 현상을 Surface Plasmon Resonance (SPR) shift effect 라고 하며, 이 SPR Shift를 UV-Visble Spectroscopy 를 통해서 확인하였다. 이런 연구를 통해 질병 메커니즘에 대한 이해를 높였고, 적용분야를 확장시켰으며, 향후 진행될 후속 연구에 도움이 될 것이다. 또한, 이러한 고유하고 특이적인 변화의 모니터링을 통해, 금 나노 입자를 새로운 detection tool 로써 이용한 간단하고 혁신적인 진단 방법의 개발 가능성을 제시하였다.

다국어 초록 (Multilingual Abstract)

Amyloidosis refers to a condition in which amyloid, an abnormal protein aggregate, accumulates in various organs, causing inflammation or causing serious disease and dysfunction. Representative amyloidosis includes Alzheimer's disease caused by aggregation of amyloid-β and Parkinson's disease caused by aggregation of α-synuclein. Amyloidosis is divided into primary amyloidosis and secondary amyloidosis. Amyloid accumulation caused by abnormal immunoglobulin production such as Alzheimer's or Parkinson's disease is referred to as primary amyloidosis and amyloid accumulation due to increased inflammatory protein production by inflammatory reaction is referred to as secondary amyloidosis. Serum amyloid A is a type of secondary amyloidosis, especially amyloid associated with rheumatic diseases. As the incidence of amyloidosis increases, research is underway on these diseases. In particular, the mechanism of formation of Serum Amyloid A fibrils in secondary amyloidosis, which has not been investigated unlike Alzheimer's disease and Parkinson's disease, is required. In this study, we observed the mechanism of formation of amyloid fibrils and the secondary structural changes. Furthermore, to meet the need for the development of a diagnostic method that can diagnose these amyloid fibrils in a simple and innovative way, we have studied and developed a diagnostic method using gold nanoparticles.
Gold nanoparticles can bind well to both the hydrophilic and lipophilic part of the protein and also to the surface of the protein with the thiol group. Using these properties, we determined that the gold nanoparticles would interact well on the surface of the amyloid fibrils, and they were expected to be useful as a detection tool. Three types of gold nanoparticles were used: a spherical shape with a diameter of 15 nm and a diameter of 50 nm, and a bar shape. These three different types of gold nanoparticles were used to simultaneously identify differences in detection by size and detection by shape.
Serum Amyloid A fibrils formation mechanism and protein secondary structure were analyzed by AFM, CD Spectroscopy and ThT Assay. The binding of the formed amyloid fibrils to three different types of gold nanoparticles was confirmed by TEM and UV-Visible spectroscopy was used to identify changes in the gold nanoparticles interacting with the amyloid fibrils. When the protein interacts with the gold nanoparticles, unique electrons distribution on the surface of the particle were changed. This phenomenon is called the surface plasmon resonance (SPR) shift effect. The SPR shift was confirmed by UV-Visble spectroscopy.
These studies have improved understanding of disease mechanisms, expanded the application area, and will be useful for further research in the future. In addition, through the monitoring of these unique and specific changes, the possibility of developing a simple and innovative diagnostic method using gold nanoparticles as a new detection tool is proposed.

목차 (Table of Contents)

• 국문초록 •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••ⅰ
• 목 차 ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••ⅲ
• List of Figure ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••ⅴ
• List of Abbreviations •••••••••••••••••••••••••••••••••••••••••••••••••••••••••ⅶ
• Ⅰ. 서 론 ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••1
• Ⅱ. 실 험 ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••5
• 1. 물질 •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••5
• 2. Serum amyloid A 섬유질 형성 ••••••••••••••••••••••••••••••••••••••••••••5
• 3. UV−Visible(UV-Vis) 흡수 스펙트럼 측정 •••••••••••••••••••••••••••••6
• 4. Circular Dichroism (CD) 분광법•••••••••••••••••••••••••••••••••••••••••••6
• 5. Thioflavin T (ThT) 형광 에세이 •••••••••••••••••••••••••••••••••••••••••6
• 6. Atomic Forced Microscopy (AFM)•••••••••••••••••••••••••••••••••••••••7
• 7. CTAB-capped Gold Nano Rod (GNR) 의 합성 •••••••••••••••••••••••8
• 8. Citrate-capped-Gold Nano Sphere (GNS) 의 합성 •••••••••••••••••9
• 9. SAA 섬유질과 금 나노 입자와의 상호작용••••••••••••••••••••••••••••••9
• 10. Transmission Electron Microscopy (TEM) 분석••••••••••••••••••10
• Ⅲ. 결 과•••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••11
• 1. 농도에 따른 SAA 섬유질 패턴 확인••••••••••••••••••••••••••••••••••••11
• 2. 시간에 따른 SAA 섬유질 형성 모니터링•••••••••••••••••••••••••••••••15
• 3. SAA와 상호작용한 금 나노 입자의 SPR shift 확인••••••••••••••••••22
• Ⅳ. 고 찰•••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••28
• Ⅴ. 결 론•••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••34
• Ⅵ. 참고문헌•••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••35
• Abstract••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••49