청대 유래의 Indirubin을 이용한 LPS로 유도된 Macrophage의 활성 조절에 관한 연구 = A Study of Indirubin from Polygonum tinctorium on the LPS-induced Macrophage Activity

다국어 초록 (Multilingual Abstract)

Rhinitis refers to an inflammatory disease of the mucous membrane covering the nasal passages. Indirubin is widely known is traditional Chinese medicine for its anti-inflammatory and anti-psoriasis effcts. The cheongdae cream developed in this laboratory has recently shown improvement in thinitis, and through this, we intend to study the rhinitis improvement effect of indirubin. Rhinitis is caused by various causes, mainly due to chronic inflammation and immune hypersensitivity. The purpose of this experiment was to find out whether indirubin had an improvement effect on immune hypersensitivity or chronic inflammation through this experiment. First, the improvement effect on chronic inflammation was confirmed. Macrophages are mainly involved in chronic inflammation, and an in vitro experiment was conducted using macrophages. Prior to the In vitro experiment, when Indirubin was treated in a water soluble medium, the problem of precipitation was solved first and the experiment was conducted. In order to increase the solubility of indirubin, pH adjustment and 1% Lysine and DMSO were used to increase the solubility. As a result, many problems of precipitation in DMSO were solved. Raw264.7 was treated with Indirubin standard and Cheongdae extract and confirmed by PCR, the mRNA expression levels of TNF-αand IL-1β were decreased. Indirubin has an inhibitory effect on the expression of IL-1β mRNA and in the case of Cheongdae extract, it has an inhibitory effect on the expression of TNF-α. Indirubin and Cheongdae extract are expected to inhibit pro-inflammatory cytokines and the mechanism of inhibition appears to be different.

목차 (Table of Contents)

• Ⅰ. 서론 1
• 1. Indirubin이 가지는 염증 억제 효과 1
• 2. 비염과 염증과의 연관성 4
• 3. 염증반응에서의 Macrophage 역할 7
• 4. 본 연구의 필요성 10
• Ⅱ. 연구 개요 11
• 1. 청대로부터의 Indirubin 추출과 세포 처리를 위한 가용화 연구 11
• 2. LPS에 의해 유도되는 Macrophage의 염증 관련 모델 설정 12
• 3. LPS에 의해 유도되는 Macrophage 활성화에 미치는 Indirubin 효과 확인 13
• Ⅲ. 실험 방법 14
• 1. 실험 방법 14
• 1.1 청대로부터의 Indirubin 추출 14
• 1.2 세포 처리를 위한 청대 추출물의 전처리 과정 16
• 1.3 청대로부터 추출한 Indirubin 가용화 방법 17
• 1.4 HPLC를 이용한 Indirubin 함량 분석 19
• 1.5 Macrophage 배양 21
• 1.5.1 Indirubin이 세포 생존에 미치는 영향 21
• 1.6 농도별 LPS로 활성화된 Raw264.7 morphology 확인 실험 22
• 1.7 LPS로 활성화된 M1 macrophage에서 분비되는 pro-inflammatory cyokines에 대한 Indirubin 효과 확인 실험 23
• 1.7.1 LPS로 활성화된 Raw264.7의 morphology 변화 비교 23
• 1.7.2 Real time polymerase chain reaction (qPCR) 24
• 1.8 통계처리 26
• Ⅳ. 연구 결과 27
• 1. Indirubin 용해도 개선 / 측정 함량 분석 27
• 1.1 pH 조절을 통한 Indirubin 용해도 개선 27
• 1.1.1 Acetic acid, glacial를 통한 pH 조절 27
• 1.1.2 Potassium Phosphate buffer를 통한 pH 조절 27
• 1.2 1% Lysine, DMSO를 통한 Indirubin 용해도 개선 29
• 1.2.1 1% Lysine을 통한 Indirubin 용해도 개선 29
• 1.2.2 DMSO를 통한 Indirubin 용해도 개선 29
• 2. Indirubin 세포 독성 30
• 3. 농도별 LPS로 활성화된 Raw264.7 morphology 확인 결과 33
• 4. LPS로 활성화된 M1 macrophage에서 분비되는 pro-inflammatory cytokines에 대한 Indirubin 효과 확인 결과 35
• 4.1 LPS로 활성화된 Raw264.7의 morphology 확인 결과 35
• 4.2 LPS로 활성화된 Raw264.7의 cytokines mRNA 변화량 측정 37
• Ⅴ. Reference 43
• ABSTRACT 46