[보고서]피폭손상융합평가 및 재생기술개발

박선후

[국가R&D연구보고서] 피폭손상융합평가 및 재생기술개발
Multidisciplinary assessment in radiation injury and application of regenerative medicine 원문보기

보고서 정보
주관연구기관	한국원자력의학원
연구책임자	박선후
참여연구자	이승숙 , 진영우 , 이효락 , 이진경 , 조민수 , 명재경 , 신동엽 , 유지영 , 장원석 , 그외 다수
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2015-01
과제시작연도	2014
주관부처	미래창조과학부 KA
연구관리전문기관	한국원자력의학원 Korea Institute of Radiological & Medical Sciences
등록번호	TRKO201500007991
과제고유번호	1711021870
DB 구축일자	2015-06-27
키워드	피폭손상,융합평가,방사선증후군,물리적 팬텀,역분화 줄기세포,3차원 organoid 배양Radiation Exposure,Acute Radiation Syndrome,Multidisciplinary assessment,Physical Phantom,iPSC,3-D organoid culture

초록 ▼

본 연구는 고선량 방사선 손상 대비 피폭손상 평가/치료/재생의료의 핵심원천 기술 개발로서,
1. 피폭선량의 평가는 피폭환자의 예후를 판단하고 치료계획을 수립하는데 있어 핵심적인 요소임. 본 연구에서는 인체와 생리학적/해부학적으로 유사한 미니돼지 physical 팬텀을 제작하였으며, 방사선 민감 장기 세포들(정상/줄기세포)에서는 방사선 선량별 유전자 손상 데이터베이스를 구축함으로써, 물리 및 생물학 분야에서 인체 방사선 손상평가 기술개발을 위한 기반을 구축하였음. 이는 향후 보다 정확한 피폭손상평가를 위한 융합 평가 시스템 개발에 활용 될 것임.
2. 고선량 피폭에 의한 급성 방사선 증후군은 증상의 다양성 및 심각성에도 불구하고 명확한 치료법의 정립 및 신의료 기술 접목이 미흡함에 따라 이를 극복하기 위해 방사선 손상 표적 장기 (폐, 골수, 위장관, 줄기세포)의 생물학적 손상 기전에 근거한 치료법의 재정립 및 표적 치료물질의 발굴이 필요함. 방사선 유도 폐섬유화 세포모델 구축 및 매개 신호전달인자 탐색, 조혈계 세포 및 위장관 장상피 세포들의 3차원 배양법을 확립 및 방사선 반응 기전 연구를 위한 모델을 구축하였음. 이는 향후 피폭증후군 장기 특이적 피폭 손상조절 인자 탐색에 적용하여 세포기반 표적치료제 개발에 활용 될 것임.
3. 현재 줄기세포치료 및 체세포 역분화 기법을 통한 인체유래 역분화 줄기세포 연구가 손상/재생의학 분야에서 급부상하고 있으나 아직 피폭손상 치료에는 접목되지 않은 실정임. 인체유래체세포에 체세포 역분화 기법을 도입하여 인체유래 역분화 줄기세포를 제작하였으며, 형태학적 및 줄기세포 표지자 염색을 통하여 생성된 역분화 줄기세포를 확인하였음. 향후 역분화 줄기세포 검증시스템을 통해 확립된 세포주는 피폭환자 맞춤형 줄기세포 치료제 개발에 활용 될 것임.
본 연구 결과물은 고선량 피폭시 피폭손상 평가기술, 피폭손상 기전 기반 피폭치료제 및 재생의료 개발을 위한 원천기술을 제시함으로서, “선량평가 선진화와 피폭환자 치료 효율 극대화 기술 개발"이라는 기관 고유 과제의 목적에 부합되는 것으로 생각됨.

Abstract ▼

IV. Results
* Current research project aims to improve the diagnostic and therapeutic techniques for radiation exposed-patient by multidisciplinary assessment in radiation injury, application of regenerative medicine and development of new target agents for radiation induced cell damage.
A. Research on the development of Multidisciplinary assessment in radiation injury
1) We developed a mini-pig physical phantom to be used in conjunction with an incorporated dosimetry system to calculate individual organ doses and effective doses.
2) We have established biological assessment system for radiation injury in radiation sensitive organs and stem cells : DNA and chromosomal damage, generation of ROS
B. Research on the modelling of ex vivo 3D culture system
We have established not only cellular model for lung fibrosis induced by radiation but also ex vivo 3D culture system of hematopoietic lineage cells and intestine cells, mimicking in vivo environment.
C. Research on development of iPSC generation system
We have generated iPSC (induced pluripotent stem cells) from human somatic cells(newborn dermal fibroblast) using retrovirus system with reprogramming factors.

목차 Contents

표지 ... 1
최 종 보 고 서 ... 2
제 출 문 ... 3
보고서 초록 ... 4
요 약 문 ... 5
SUMMARY ... 11
CONTENTS ... 13
목 차 ... 14
제 1 장 연구개발과제의 개요 ... 15
제 1절. 연구 개발의 목적 및 필요성 ... 15
제 2 절. 연구의 범위 ... 15
제2장 국내외 기술개발 현황 ... 17
제 1절. 선진국 연구개발 동향 및 기술수준 ... 17
제 2 절. 국내 연구개발 동향 및 선진국과의 기술격차 ... 19
제 3 장 연구 개발 수행 내용 및 결과 ... 21
제 1 절 물리적 손상평가시스템 설계 및 방사선 민감장기 세포특이적 선량별 평가 시스템 탐색 ... 21
제 2 절 피폭증후군 표적장기 세포들 (폐, 골수, 위장관)의 방사선 반응모델 및 손상 신호경로 탐색 ... 32
제 3 절 인체유래 역분화줄기세포 생성 및 생성효율에 대한 방사선의 영향 ... 46
제 4 장 목표달성도 및 관련분야에의 기여도 ... 65
가. 최종 목표 ... 65
나. 연도별 연구개발 목표 및 달성도 ... 65
제5장 연구 개발결과의 활용 계획 ... 67
제 1절 기술적 측면 ... 67
제 2절 사회ㆍ경제적 측면 ... 67
제 3절 기타 ... 68
제6장 연구개발과정에서 수집한 해외과학기술정보 ... 69
제 7 장 참고문헌 ... 71
끝페이지 ... 74

표/그림 (25)

표 Conversion from CT images to 3D STL image data. 2D medical images were acquired from a tomograph at Ian animal diagnostic imaging center. The CT images are 2D slices saved in standardized format DICOM and those were used as the input data in this project. By performing 3D image processing, selected Hounsfield value or gray scale value of ROI(Region Of Interest) can provide 3-dimensional geometry information.
표 Design of mini-pig physical phantom. The mini-pig physical phantom is constructed on a slice-by-slice basis with a resolution of 2 mm, and holes for dosimeters(alanine dosimeter, glass dosimeter) were considered into slices prior to assembly. Thus the slices will be fixed together to form the complete phantom. The phantom will be used in conjunction with a complementary dosimetry system which is using two different dosimeters to measure individual organ doses.
표 Alanine EPR spectrum(left) and dose response curve of alanine dosimeter(right). 20 Gy of radiation was irradiated for the alanine sample to obtain the alanine spectrum by using EPR equipment. Besides dose response curve was compiled after radiation exposure experiments on five samples at each particular dose range. The curve showed good linearity even at low doses range(coefficient of determination R2 = 0.9999).
표 Analysis of chromosomal damage and DNA damage in various normal and stem cells in radiation response. Various cells replate and exposed to radiation in manner of indicated dose of radiation. At 1 hour or 24 hours after irradiation, DNA damage was analyzed by p-H2AX and micronuclear(MN) formation was evaluated using confocal microscope.
표 Analysis of cell death and radiation related ROS generation in various normal and stem cells. Cells showing various sensitivity were replated and exposed to radiation in manner of indicated dose. Cell death, loss of mitochondria membrane potential (MMP) and reactive oxygen species were analyzed.
표 Analysis of radiation induced cell death of lymphocyte isolated from mouse or rat. Lymphocytes and bone marrow cells were harvested from mouse or rat at 24 hours and 48 hours after irradiation. Cell death was analyzed by trypan blue staining.
표 Analysis of cellular senescence after irradiation in various normal and stem cells using ß-gal staining. Various cells were replated and exposed to radiation in manner of indicated dose of radiation. After 5 day, cellular senescence was analyzed by senescence-associated (SA) ß-gal staining kit.
표 Expression of SMA in tissue from mini-pig exposed by radiation. H&E staining reveals prominent fibrotic bands along the interlobular septums. Type 1& 2 pneumocytes reveals no immunostaining for SMA. Activated fibroblasts in fibrotic bands reveals diffuse and strong immunoreactivity for SMA
표 Enhanced markers of lung fibrosis in WI38 cells (Fibroblast cell line) in response to radiation. TGFβ1 concentration in the supernatants of confluent WI38 cells, cultured with or without 5Gy irradiation as indicated dose. TGFβ1 and synthesis of collagen was measured by ELISA. At the time of harvest, cells were counted and the TGFβ1 and synthesis of collagen values were corrected as proportion to cell numbers. Indicated protein expression was analyzed by western blotting.
표 Analysis of signaling factors associated with lung fibrosis induction in BEAS2B, WI38 and A549 cells in response to radiation. Various lung epithelial cells were exposed with or without 5 Gy radiation. After 3 days, cells were harvested. Indicated protein expression was analyzed by western blotting.
표 Cell death induced by radiation in jurket cells and induction of p53 and PUMA expression. Jurket cells were exposed with or without 1, 2, 4 Gy radiation. After day 3, cell death were analyzed by flow cytometry and indicated protein expression was analyzed by western blotting.
표 Ex vivo culture system of hematopoietic lineage cells (Jurket and Mo7e) using alginate mixture. Small colonies of hematopoietic cells (Jurket and Mo7e) could be seen from day 5, and colonies grew larger and more numerous as the culture time was prolonged.
표 Analysis of cell death and signaling pathway induced by radiation in normal intestine cell line (InEpC) and tumor intestinal cell line (Caco2). Intestine cells (InEpC and Caco2) were exposed with or without up 30 Gy radiation. After 3 days, cell death were analyzed by flow cytometry, expression of gene associated hedgehog signaling and wnt signaling pathway was performed by RT-PCR and indicated protein expression was analyzed by western blotting.
표 Scheme of ex vivo culture of crypt isolated from mouse and mophology of isolated crypt from mouse. (A) Methods of 3D organoid culture using matrigel from isolated crypt of C57/B6 mice small intestin. (B) Image of isolated crypt from small intestin of C57/B6 mice.
표 Organoid formation in 3D culture system. Bright-field images of small-intestinal crypts in ENR-CV culture system : EGF, Noggin and R-spondin 1 (ENR) media containing 3uM CHIR99021, 1mM valproic acid.
표 Efficiency of GFP-retrovirus infection in NUFF and expression of reprogramming 4 factors (Klf4, Oct4, Sox2, c-Myc). The cells were replated and infected with GFP-retrovirus. After 5 days, the expression of GFP was compared by flowcytometry. (A) The percentage of GFP expression cells after infection (B) The expression of Klf4, Sox2, Oct4 and c-Myc was analyzed by western blotting. Actin was used as loading control. (C) The morphology of transduced NUFF at day 5.
표 Morphology and surface marker expression of transduced human NUFF cell colonies. Transduced NUFF cells were cultured on MEF feeder system with hES medium. Morphology(left) and live-staining of Tra1-81(right), surface marker of pluripotent stem cells.
표 Expression of other surface markers(SSEA4, Tra1-60) of pluripotent stem cells in subcultured NUFF-hiPSC colonies. iPSC colonies were picked up and sub-cultured. Images of sub-cultured iPSC colonies and expression of SSEA4, Tra1-60 and alkaline phosphatase (AP) staining at passage 4. Medium was changed daily. Bars indicate 200 um.
표 Efficiency of GFP-retrovirus infection and expression of reprogramming factors (Klf4, Oct4, Sox2, c-Myc) in radiation exposed NUFF cells with various dose. The cells were replated and infected with GFP-retrovirus. After 5 days, the expression of GFP was compared by flowcytometry. (A) The percentage of GFP expression after infection (B) The expression of Klf4, Sox2, Oct4 and c-Myc was analyzed by western blotting. Actin was used as loading control.
표 Analysis of reprogrammed colonies by AP assay. iPSC colonies were stained with alkaline phosphatase assay kit at week 3. Images of stained iPSC colonies and quantification of numbers of stained colonies.
표 Multipotent fibroblast from mouse newborn skin. Dermal fibroblasts were isolated from back skin of newborn of C57B/B6 mice. (A) Immunophenotype of skin fibroblast at passage 3 was analyzed by flowcytometry. Black line indicates to isotypic controls and grey surface presents stained cell numbers with the indicated antibodies. (B) Immunofluorescent staining of isolated dermal fibroblast. Cells were stained for Fibronectin and alpha-smooth muscle actin (fibroblast markers) as well as vimentin and nestin (multipotent fibroblast markers), respectively. Nuclei also were stained with DAPI. Bars, 50 μm. Green color represents indicated antibody. Blue color is counterstaining to nuclei.
표 Reduced expression of CD105 with passage dependent manner in mouse dermal fibroblast. Isolated cells were passed continuously and the expression of surface antigens of cells were analyzed by flowcytometry at indicated passage.
표 Characterization of mouse dermal fibroblast with CD105high or CD105low. Cultured dermal fibroblast (passage 3) were stained with PE-CD105, a cell surface marker of MSC. CD105 positive cells were separated using MACS PE-conjugated MicroBeads followed by FACS analysis. (A) Representative plots of cells stained with PE-CD105 before, after flowthrough of MACS sorting. (B) Immunophenotype of purified cells was detected by flow cytometry at passage 5. (C) The comparison of growth rate of purified cells. Purified cells were seed as same density and countered by tryphan blue staining after incubation for indicated time.
표 Enhanced adipogenic and osteogenic differentiation potential of mouse dermal fibroblast expressing CD105. Purified cells with CD105 were cultured in differentiation inducing medium for 3 weeks. (A) adipogenic induction was appeared by accumulation of lipid-rich vacuoles as judged by staining with Oil Red O (original magnification x 200) in both cells. (B) RT-PCR shows the enhanced PPARγ mRNA expression in CD105 positive cells. (C) Calcium deposition was detected by Alizarin Red in cells without CD105 (top pannel) or with CD105 (bottom pannel). Original magnification is x 100. (D) RT-PCR detecs the increased mRNA expression of alkaline phosphatase (ALP) and Runx2 in CD105 positive cells. GAPDH used to internal control.
표 Primer sequences used in RT-PCR analysis

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[국가R&D연구보고서] 피폭손상융합평가 및 재생기술개발
Multidisciplinary assessment in radiation injury and application of regenerative medicine 원문보기