[보고서]당화된 고밀도지단백질 (HDL)의 노화 유발 및 HDL 기능 강화로 노화 개선

[국가R&D연구보고서] 당화된 고밀도지단백질 (HDL)의 노화 유발 및 HDL 기능 강화로 노화 개선
Induction of senescence by glycated high-density lipoproteins (HDL) and Prevention of aging by enhanced HDL 원문보기

보고서 정보
주관연구기관	영남대학교 YeungNam University
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2014-04
과제시작연도	2013
주관부처	미래창조과학부 Ministry of Science, ICT and Future Planning
과제관리전문기관	한국연구재단 National Research Foundation of Korea
등록번호	TRKO201500003944
과제고유번호	1711005886
사업명	중견연구자지원
DB 구축일자	2015-05-23
키워드	고밀도지단백질.아포지단백질.당화.산화.제브라피쉬.조직재생.라파마이신.세포노화.염증.high-density lipoproteins.apolipoproteins.glycation.oxidation.zebrafish.rejuvenation.rapamycin.senescence.inflammation.
DOI	https://doi.org/10.23000/TRKO201500003944

초록 ▼

연구의 목적 및 내용
최종목표: 변형된 HDL 검출을 통한 노화 조기 진단 및 노화와 당뇨 관련 질환 억제를 위해 기능이 강화된 HDL의 합성과 평가
• 노화스트레스 (당화, 산화)에 의한 지단백질 변형 검출,
• 노화관련질환 (당뇨, 동맥경화) 조기 검출 및 합병증 억제 단백질 개발
• 고밀도지단백질을 이용한 난용성 약물 전달체/바이러스유전자 전달체 개발
• 생활환경제품 성분 독성 연구: 노화유발인자로서 가습기살균제의 작용원인 규명
• 당화가 HDL의 변형으로 동맥경화와 당뇨를 악화시키는 메카니즘 규명
• 지단백질의 변형이 세포 노화 조직손상에 관여하는 메카니즘 연구하고,
• 기능이 강화된 재조합 HDL의 합성으로 노화스트레스를 억제한다
• 노화와 당뇨 및 동맥경화의 유발 상관성을 혈청 지단백질의 변형 관점에서 설명
연구결과
• V156K-apoA-I 고밀도지단백질을 이용한 당뇨성 족부궤양 치료제 개발
• 고밀도지단백질들의 인슐린 분비 촉진 기능 비교,
• V156K-apoA-I 고밀도지단백질을 이용한 난용성 약물 전달체 개발 (국내특허등록 및 미국, 중국 특허 출원
• 라파마이신, 레스베라트롤, 커큐민등의 수명연장물질들의 용해도 증가 혈관 대식세포,피부섬유세포, 제브라피쉬 배아와 성체를 이용한 전달능 향상
• 가습기 살균제 성분 (PGH, PHMG)의 지단백질 변형 및 심혈관독성 연구
• 살균제 성분의 혈관세포모델에서 산화, 염증기능 비교, 동맥경화 유발 및 피부세포노화 유발 비교, 제브라피쉬 배아독성 및 성체 노출시 급성 심혈관 독성, 지방간 관찰,
• 노화스트레스에 의해 변형된 HDL의 리피드와 단백질 성상 및 기능 변화 관찰: 노인혈청 HDL 과 청년혈청 HDL 비교 (진단 방법 특허 등록).
• 노화 스트레스에 의해 변형된 HDL의 분리 검출 및 세포 활성 비교
• 제브라피쉬 배아 모델에서 변형된 HDL의 염증 유발 및 노화 유발 비교 (상위 2% 논문 게재)
• 변형된 HDL의 검출로 노화진단마커 개발 및 등전점 전기영동으로 노화진단 (상위18% 논문게재)
• 트란스지방에 의한 HDL 의 변형과 동맥경화 유발 및 배아 독성 메카니즘 규명 (상위 1% 논문 게재)
• 3년간의 연구를 통해 SCI 국제학술지 9편, 국내특허 등록 8건, 국제출원 2건, 석사11명 배출하는 우수한 성과를 거두었다.
연구결과의 활용계획
• 변형된 HDL 검출을 통한 노화 및 노화 관련 질환 조기진단으로, 대사증후군, 심혈관질환 등을 예방 및 치료에 활용
• 라파마이신등을 HDL 수용액상에 녹여 전달하는 약물전달 방법 개발로 기타의 난용성 약물 치료제 범위 확대에 활용
• 지단백질/아포지단백질을 이용한 바이오메디칼 산업 응용은 다양한 약물전달체, 유전자 전달체로의 용용이 가능하므로, 기술이전 등 산업적 제품화.

Abstract ▼

Purpose&contents
Investigation of exacerbation mechanism of diabetes and atherosclerosis by glycated high-density lipoproteins (HDL) including cellular senescence and tissue necrosis as diabetic complications. Suppression of aging stress and induction of tissue regeneration using enhanced HDL containing V156K and rapamycin.
• Explanation of incidental relationships among aging, diabetes, atherosclerosis in viewpoint of lipoprotein modification
• Production of a new reconstituted HDL, which has more insulin secretion activity and resistant to the glycation.
• Establishment of zebrafish model for senescence, diabetes, atherosclerosis including their complications such as tissue damage and regeneration
Result
• Detection of modified high-density lipoproteins (HDL) from aging process
• Cellular activity of elderly or modified lipoproteins in senescence and inflammation
• Comparison of resistance against glycation stress between WT and V156K
• Analysis of reconstituted HDL by glycated WT and V156K using electroproperties
• Identification of aging biomarkers in HDL by glycation stress
• Anti-senescence and anti-atherosclerotic activity of V156K-apoA-I, and its resistance against glycation
• Insulin secretion stimulation in pancreatic β-cell (INS-1) cell by V156K-rHDL
• Establishment of zebrafish model for atherosclerosis and diabetes model
• Encapsulation of rapamycin in HDL and evaluation of therapeutic effect.
• Analysis of anti-senescence effect of rapamycin in V156K-rHDL
• Induction of tissue rejuvenation and regenerationon by V156K-rHDL in diabetic zebrafish
• Exacerbation of cellular senescence and atherosclerosis by trans fatty acid
• Embryotoxicity of glycated apoA-I and HDL in zebrafish
• Cardiovascular toxicity of PGH and PHMG via modification of lipoprotein
Expected Contribution
• Application for development of anti-diabetic and anti-aging agents, early detection of aging and aging-related disease
• Application for development of plaque removal and blood circulation
• Application of drug-delivery vehicle using recombinant HDL for nonsoluble agents such as rapamycin
• With potent anti-aging and anti-atherosclerotic effects of rHDL, biosimilar drugs can be developed.

목차 Contents

핵심연구사업 최종보고서(평가용) ... 1
목차 ... 3
연구계획 요약문 ... 4
연구결과 요약문 ... 5
SUMMARY ... 6
연구내용 및 결과 ... 7
1. 연구개발과제의 개요 ... 7
2. 국내외 기술개발 현황 ... 11
3. 연구수행 내용 및 결과 ... 13
4. 목표달성도 및 관련분야에의 기여도 ... 36
5. 연구결과의 활용계획 ... 38
6. 연구과정에서 수집한 해외과학기술정보 ... 39
7. 주관연구책임자 대표적 연구실적 ... 41
8. 참고문헌 ... 43
9. 연구성과 ... 44
10. 기타사항 ... 69
끝페이지 ... 87

표/그림 (23)

표 Reactive oxygen species (ROS) induced damage in diabetic endothelial cells. Reactive oxygen species-induced DNA strand breaks induce upregulation of poly(ADP ribose) polymerase (PARP) that in turn inhibits glyceraldehyde phosphate dehydrogenase (GAPDH). AGE receptors activate intracellular signalling cascades leading to apoptosis through inhibition of PI3K/Akt/eNOS signalling and activation of the NF-κB and c-Jun NH2-terminal kinase (JNK) pathways as well as NADPH oxidase-dependent ROS generation.
표 Synergistic effect of ROS and AGE in diabetes and atherosclerosis.
표 지단백질의 분류. HDL, high-density lipoprotein (고밀도지단백질); LDL, low-density lipoprotein (저밀도지단백질); VLDL, very-low density lipoprotein (최저밀도지단백질)
표 Beneficial effect of HDL (left) and quality balance of HDL to maintain its beneficial effect
표 A. Electrophoretic patterns of native apoA-I and glycated apoA-I (15% SDS-PAGE). Mixture of apoA-I (2 mg/mL) and fructose (final concentration, 5 mM) was incubated for 90 h under 5% CO2at37oC. After protein determination, 5 mg of protein was loaded per lane. Lane 1, native apoA-I; lane 2, glycated apoA-I; lane M, molecular weight marker (BioRad, low range standard). B. Survival of zebrafish embryos which were injected with LPS plus nA-I-rHDL or gA-I-rHDL on day 1 post-fertilization. As a control, LPS (200 ng) alone and each rHDL alone (50 ng) were also injected into the embryo. Prior to injection, LPS (200 ng) was mixed with 50 ng of nA-I-rHDL or gA-I-rHDL. *, p <0.05; **, p <0.01
표 Survival of zebrafish embryos after co-injection of oxLDL (13 ng of protein) plus vitamin C (final concentration, 1 mM) or each rHDL (final concentration, 1 mM). PBS, phosphate buffered saline; *, p< 0.05;**,p< 0.01
표 Fluorescence image (Ex=588 nm, Em=605 nm) of zebrafish embryo was obtained using a Nikon Eclipse TE2000 microscope (Tokyo, Japan) after dihydroethidium (DHE) staining as described in the text.
표 Determination of the extent of glycation in a time-dependent manner
표 Antioxidant ability of WT and V156K apoA-I in the native and glycated states against cupric ion-mediated oxidation of LDL.
표 Insulin secretion activity of apoA-I in lipid free (panel A) and lipid-bound (panel B) state, under native (n) or glycated (g) condition. Rat insulinoma cells (INS-1) were incubated for 2 hr in the presence of apolipoproteins (final 1 mM in the media) at different glucose concentrations (final concentrations 2.8 or 25 mM in culture medium). Insulin levels in the medium were quantified using a radioimmunoassay kit. The results are expressed as the mean ±SD from three independent experiments with duplicate samples. *, p <0.05;**,p <0.01compared with its native state.
표 Intramural injection of Ad alone or PL-Ad into Balb/c nude mice, which was established as the Hep3B tumor xenograft model. The same amount of Ad (3 ×109 virus particles) was injected three times as indicated by the arrow ,and tumo r volume was measured at 34 dayspost-injection.
표 Histological analysis of tumor area at 3 days post final injection by hematoxylin and eosin (H & E) staining. Particles of adenovirus (Ad) in the tumor area were visualized by immunohistochemistry using the antiAd E1A antibody. Proliferating cell nuclear antigen (PCNA) was visualized by immunodetection using mouse anti-PCNA antibody
표 Immunodetection of apolipoproteins and enzymes in HDL3 between the AP and control groups. An equal amount of HDL3 (6mg of protein) from an individual subject was loaded per lane. Polyclonal CETP antibody (abcam, ab19012), apoC-III antibody (Chemicon, AB821), and LCAT antibody (abcam, ab786) were used as primary antibodies.
표 Comparison of the antioxidant activity of HDL.
표 Properties of HDL2 from the AP and control groups. Susceptibility to oxidation in HDL2 by cupricion treatment (graph). Representative picture of negatively-stained HDL2 from the angina pectoris (AP) and control groups (electronmicroscopy). All micrographs are shown at a magnification of 40,000x. The scale bar corresponds to 100 nm. MDA, malondialdehyde. #, p <0.01versus control under presence of cupric ion.
표 Denatured electrophoresis profiles of human HDL. HDL was incubated with the disinfectants for 1hr and subjected to 15% SDS-PAGE. Each lane contains 3 μg of protein. Lane B, apoA-I alone; lane 1, H2O-treated;lane 2, 0.3% PHMG treated; lane 3, 10 mM EDEA-treated; lane 4, 10 mM Gnd-HCl-treated ; lane 5, 10 mM PGH (10 mM EDEA + 10 mM Gnd-HCl) treated.
표 Modification of human LDL by the sterilizers
표 Uptake of oxidized LDL (oxLDL) in the presence of disinfectants. Differentiated THP-1 cells were incubated with 50 mL of oxLDL (50 mg of protein) in the presence of each disinfectant for 48 hr. a, PBS alone; b, oxLDL+ PBS; c, oxLDL + 0.03% PHMG (final); d, oxLDL + 1 mM EDEA (final); e, oxLDL + 1 mM Gnd-HCl (final) ; f, oxLDL + 1 mM PGH (final, EDEA + Gnd-HCl). A. Increased cellular uptake of oxLDL as visualized by oil-red O staining. B. Oil-red O-stained cellular areas of 0.8 mm2 were quantified using computer-assisted
표 Acceleration of cellular senescence by disinfectants in human dermal fibroblasts (HDFs). Representative image of SA-β-gal-positive HDFs (blue staining). Cells were treated with PHMG (final concentration of 0.03%) or PGH (final concentration of 1 mM) from passages 9-13 and analyzed as described in the Methods. Cell images were captured using a Nikon Eclipse TE2000 microscope (Tokyo, Japan) at 100x magnification. Lower numbers indicate cell numbers counted at passage 13. Inset graph shows percentage of SA-β-gal-positive cells per 7.4 mm2ofcell-cultureareaduringtreatmentthesterilizer. Data are shown as the mean±SD of three independent experiments performed in duplicate. **, p<0.01;***,p<0.001.
표 Change in embryo stage and ROS production after injection of disinfectants at the designated times. Both PHMG- and PGH-injected embryos showed attenuated development speed along with greater ROS production. Fluorescence image (Ex=588 nm, Em=605 nm) of zebrafish embryos was obtained using a Nikon Eclipse TE2000 microscope (Tokyo, Japan) after dihydroethidium (DHE) staining as described in the text.
표 Survival graph of zebrafish in water containing disinfectant. Acute deaths were observed in water containing PHMG or PGH. A. Swimming patterns of zebrafish in water containing PHMG (final concentration of 0.3%). B. Swimming patterns of zebrafish in water containing PGH (final concentration of 10 mM).
표 Histologic analysis of longitudinally cross-sectioned bulbous artery and heart following death by exposure to disinfectant solution. All images were obtained at 400x magnification. a. Hematoxylin & Eosin staining b. Masson’s trichrome staining to visualize collagen fibrosis and accumulation. c. DHE staining.
표 트랜스지방 급여에 의한 제브라피쉬의 지방간 및 간 염증 유발 악화

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연합인증

[국가R&D연구보고서] 당화된 고밀도지단백질 (HDL)의 노화 유발 및 HDL 기능 강화로 노화 개선
Induction of senescence by glycated high-density lipoproteins (HDL) and Prevention of aging by enhanced HDL 원문보기