골수 중배엽 줄기 세포의 노화가 BMP-2에 의한 줄기 세포 분화에 미치는 영향

학위논문 (박사)-- 서울대학교 대학원 : 의과대학 의학과, 2018. 2. 이춘기.Background: Mesenchymal stem cells (MSCs) are cells having potential to differentiate various type of cells including osteoblasts, adipocytes, or chondrocytes. Bone morphogenetic protein-2 (BMP-2) is considered as a powerful inducer to promote cascade of differentiation into osteoblasts. Recently, BMP-2 has been tried as a bone graft substitute when spinal fusion surgery is performed in old age patients. However, lack of knowledge about the differentiation of MSCs by BMP-2 and the effect of aging has been one of problems for extensive use of BMP-2. In this regard, we intended to compare the effect of recombinant human BMP-2 (rhBMP-2) on differentiation of MSCs into osteoblasts and adipocytes between aging MSCs and young (control) MSCs. Materials and methods: Cellular aging (senescence) was induced by replicative passages with cultures under high level glucose. Senescence-associated ß-galactosidase (SA-ß-gal) staining, and senescence related markers (p16, p21, and p53) were used to confirm aging MSCs. Aging and young (control) MSCs were cultured in osteogenic and adipogenic differentiated medium with different concentration of BMP-2 (0, 25, 100, 250, and 500 ng/ml). We compared the phenotypes by Calcium assay, adipogenesis assay, and specific staining (Alkaline Phosphatase, Alizarin Red S, Oil red O), and gene expression patterns by real-time polymerase chain reaction (PCR), western blot assay, and microarray analyses between aging MSCs and control MSCs. Microarray analysis was conducted with 4 different cells (aging MSCs and control MSCs with or without the induction of BMP-2). GSEA (Gene Set Enrichment Analysis), leading-edge subset analysis, and MetaCore analysis were used as a microarray analysis. Results: Aging cells by replicative passages under high level glucose (22 mM) were well characterized by senescence markers. Osteogenic differentiation by BMP-2 decreased in aging MSCs compared to control MSCs. Lower ALP activity and less Ca++ release was observed in aging MSCs (P < 0.001, and P = 0.005, respectively), and positive dose-response relationship was observed in both MSCs. The mRNA expression of Runx-2, bone sialoprotein (BSP) and osteocalcin (OCN) was lower in aging MSCs. However, adipogenic differentiation by BMP-2 was comparable between 2 cells. Adipogenesis assay showed no differences of adipogenic potential between 2 groups (P = 0.279). In addition, there was no superiority in the expression of PPARγ between 2 cells. These results were also supported in protein level by western blot assay. Microarray analysis showed more upregulated genes in rhBMP-2-treated aging MSCs than in rhBMP-2-treated control MSCs and non-treated aging MSCs. Aging and adipogenesis related gene sets were more upregulated and significantly enriched by the induction of rhBMP-2 in aging MSCs compared to control MSCs. In BMP related leading edge subsets, 6 clustered genes (CHRDL1, NOG, SMAD1, SMAD7, FST, and BAMBI) were upregulated in aging MSCs. In aging MSCs, NF-kB or p38 MAPK pathway could be important in response to BMP-2, whereas BMP/SMAD pathway in young MSCs, based on MetaCore analysis. Conclusions: Aging MSCs showed less osteogenic differentiation and comparable adipogenic differentiation in response to BMP-2 compared to control MSCs. Relative superior adipogenic differentiation by BMP-2 in aging MSCs is supported by the result of microarray analysis. In addition, several genes might be candidates for senescence dependent BMP-2 effect on differentiation of MSCs. Different signaling pathway might play a role in different response to BMP-2 between aging and young MSCs. Further in vivo research along with the current study may contribute to effective use of BMP-2 in clinical fields by elucidating the signaling pathway whether MSCs differentiate into osteoblasts or adipocytes by BMP-2.Introduction 1 Materials and Methods 6 Results 17 Discussion 47 Conclusion 57 References 58 Abstract (Korean) 70Docto

Evaluation of Phenotypic Characteristics in Endothelial Cells Isolated from Intramuscular Hemangioma

학위논문 (석사)-- 서울대학교 대학원 : 의학과, 2012. 2. 김한수.목 적: 심부 연부 조직에서 가장 흔히 발생하는 유형인 근육내 혈관종의 발병 기전에 중요한 역할을 하는 내피세포의 분리 및 배양법을 확립하고, 근육내 혈관종 내피 세포의 특성을 규명하여 근육내 혈관종의 치료제 개발에 필요한 기초적 자료를 제공하고자 하였다. 대상 및 방법: 수술시 얻어진 9개의 근육내 혈관종 조직에서 내피 세포의 분리 및 배양을 시행하였다. 인간 심장 미세혈관 내피세포를 대조군으로 사용하였다. 분리된 내피세포의 순도는 내피세포에 특이적 표지자인 CD31에 대한 단일클론항체로 세포를 염색하여 유동 세포 계수법으로 정량분석하였다. 분리된 내피세포의 형태학적, 기능적 특성을 보기 위하여 면역화학염색 및 Matrigel 튜브 형성 분석을 각각 시행하였다. 혈관형성 조절 인자인 VEGF-VEGFR 경로와 Ang1-Tie2경로의 발현 정도를 확인하기 위하여, 실시간 PCR 검사를 하였다. 근육내 혈관종 내피세포와 인간 심장 미세혈관 내피세포에서 Ang1과 VEGF에 대한 반응으로 세포 이동능과 증식능을 확인하기 위하여, 세포 이동능 분석 및 세포 증식능 분석을 각각 시행하였다. 과발현된 Tie2의 하위 신호전달 경로를 찾기 위하여, western blot 검사를 통해 Akt, p-Akt, FOXO1, p-FOXO1의 발현을 확인하였다. Tie2-Akt-FOXO1 경로가 근육내 혈관종 내피세포에서 지속적으로 활성화되어 있는지 알아보기 위해, 6명의 근육내 혈관종 환자 조직으로부터 분리 배양한 내피 세포를 이용하여 western blot 검사를 시행하였다. 결 과: 분리된 내피세포는 광학 현미경하에서 특징적인 모양을 보였고, 유동 세포 계수법에서 높은 순도를 보여주었다. 또한 Matrigel 튜브 형성 검사에서 정상적인 내피세포의 튜브 형성 기능을 나타내었다. 인간 심장 미세혈관 내피세포에 비하여 근육내 혈관종 내피세포에서는 Tie2와 VEGFR1이 과발현됨을 확인하였다. 근육내 혈관종 내피세포에서는 Ang1에 대한 반응으로 세포 이동 및 생존 증가를 보여주었다. 이에 반해, VEGF에 대한 반응은 근육내 혈관종 내피세포보다 인간 심장 미세혈관 내피세포에서 더 확연하였다. 인간 심장 미세혈관 내피세포에서 Ang1에 의해 활성화된 Tie2는 Akt의 인산화와 함께 FOXO1의 인산화를 유도하였다. 근육내 혈관종 조직과 대조군 사이 Tie2, Akt, p-Akt, FOXO1, p-FOXO1의 발현 정도를 비교하였을 때, 모든 혈관종 조직에서 Tie2가 과발현되었고, Akt와 FOXO1의 경우 양쪽 모두에서 발현되었지만, p-Akt와 p-FOXO1의 경우는 오직 근육내 혈관종의 경우에만 발현되었다. 결 론: 근육내 혈관종에서 내피세포의 분리 및 순수화 작업은 추후 근육내 혈관종의 생물학이나 병리 기전 연구에 유용한 도구가 될 것으로 생각된다. 본 연구 결과는 근육내 혈관종 내피세포에서 Tie2 과발현이 Akt 신호를 활성화시키면서 FOXO1을 억제하는 것을 보여주었다. 이는 Tie2-Akt-FOXO1 경로가 근육내 혈관종의 발병에 있어 중요한 역할을 하리라는 것을 시사한다. 본 연구는 근육내 혈관종의 약물 개발에 기여할 것으로 기대된다.Purpose : The purpose of this study is to establish the procedure about isolation and purification of endothelial cells from intramuscular hemangioma, characterize their angiogenic phenotype and functional characteristics, and search a possible signaling pathway related to development of intramuscular hemangioma. Materials and Methods : Intramuscular hemangioma specimens were obtained from 9 patients who underwent surgical resection. Endothelial cells (ECs) from intramuscular hemangioma were isolated and cultured by digestion, filtering, washing, incubation and purification in sequence. Human cardiac microvascular endothelial cell (HCMVEC) was used as control EC. The purity of isolated ECs was quantified with flowcytometry by staining the cells with monoclonal antibody to vWF and CD31, typical markers of EC. To examine the morphological and functional characteristics of isolated ECs, immunohistochemistry and Matrigel tube forming assay were used respectively. To observe the expression of angiogenesis regulators such as VEGF-VEGFR system and Ang-Tie2 system, real time PCR was used to check relative expression levels of them. To determine the migration and proliferation activities of ECs from intramuscular hemangioma and control ECs in response to Ang1 and VEGF, cell invasion assay and cell proliferation assay were used respectively. To investigate downstream signaling pathway of overexpressed Tie2, western blot analysis was performed for detection of Akt, phosphorylated Akt (p-Akt), FOXO1, phosphorylated FOXO1 (p-FOXO1). To test if Tie2-Akt-FOXO1 pathway is constitutively activated in ECs from intramuscular hemangioma, cell lysates from 6 patients samples were analyzed with western blot analysis. Results : Isolated ECs showed typical appearance in a light microscope and high purity by flowcytometric analysis. They also showed normal tube forming function of ECs in Matrigel tube forming assay. The expression of Tie2 and VEGFR1 was up-regulated in ECs from intramuscular hemangioma compared to control ECs. Increased migration and proliferation of ECs from intramuscular hemangioma in reponse to Ang1 was more evident compared to control ECs. However, the migration and proliferation response to VEGF was more evident in control ECs. Activation of Tie2 by Ang1 induced phosphorylation of Akt accompanied by phosphorylation of FOXO1 in control ECs. Comparing the expression of Tie2, Akt, p-Akt, FOXO1 and p-FOXO1 between hemangioma samples and controls, increased Tie2 expression was confirmed in all hemangioma samples. Though Akt and FOXO1 were expressed in both, the expression of p-Akt and p-FOXO1 was only observed in hemangioma samples. Conclusion : Isolation and purification procedures of ECs from intramuscular hemangioma could be a useful tool for further research about biology and pathomechanism of intramuscular hemangioma. Our findings suggest that up-regulated Tie2 in ECs from intramuscular hemangioma activates Akt signaling with inhibition of FOXO1. These results suggest that Tie2-Akt-FOXO1 pathway may play a key role in the tumorigenesis of intramuscular hemangioma. The current study is expected to contribute to development of medical treatment of intramuscular hemangioma.Maste

열린충남 7호-[특집]21세기 한국농업의 발전전략과 미래모습

Ⅰ. 한국농업의 국내외 여건변화 70년 중반까지 농업은 공업부문에 비해 투자 우선순위에서 열위에 있었지만, 식량생산 산업으로서 그 중요성에 의문이 제기되지 않았다. 식량생산이 부족하고 외환이 부족하였던 이 시기에는 주곡을 자급하기 위한 농업생산기반 확충이 농정의 중심이었다. 그러나 70년대 중반 이후부터는 산업화의 진전으로 농·공간 생산성과 소득격차가 계속 확대되어 농가소득 문제가 정치·사회적인 문제로 등장하게 되었다. -이후 생략1. 한국농업의 국내외 여건변화 2. 21세기를 향한 한국농업의 발전전략 3. 21세기 한국농업의 미래모

유연체 시뮬레이션을 위한 적응성 질량-스프링 모델

학위논문(석사) - 한국과학기술원 : 정보통신공학과, 2009. 8., [ ix, 52 p. ]Traditional computer simulation uses only a mouse, keyboard, and monitor as the input and output devices. With the recent emergence of haptic techniques, which can give users kinetic and tactile feedback, the field of computer simulation is diversifying. In particular, the need for virtual-reality-based surgical simulation has become great, stimulating new research the practical virtual simulation of surgery. In this thesis, we present a framework for a hybrid model of simulation that can realistically simulate the behavior of the soft, pliable human body, along with haptic feedback from the user’s interaction with it. A soft body simulation algorithm, which we refer to as the adaptive mass-spring model algorithm, is the core technique of the framework we present. Our framework has both a behavior model and a haptic model to support both the deformation of an object and the haptic feedback. The behavior model uses the adaptive mass-spring algorithm to calculate the realistic deformation of a virtual object in real time. Since our purpose is to provide a virtual environment for simulated surgery, virtual organs should reflect the properties of their corresponding real organs. Each virtual organ should be based on volumetric data because each real organ contains other tissues or vessels. The traditional soft body simulation algorithm uses surface data to represent the virtual object because this data is simpler than volumetric data. Since volumetric data includes other information internal to the organ, there is great computational complexity in the volume-data-based algorithm, which presents an obstacle to the execution of soft body simulation in real time. Our framework uses the adaptive mass-spring algorithm to reduce this computational complexity. The adaptive algorithm is an efficient algorithm that distributes the resources of the framework to the various elements according to their individual amounts of energy. First, the virtual object is divided into...한국과학기술원 : 정보통신공학과

열린충남 7호-[통합본]열린충남 7호 (1997, 봄)

[특집]충남농업의 VISION과 대응전략 충남농업의 어제와 오늘 _ 이종상 Ⅰ. 서론 최근 수년간 한국농업은 국내외적으로 많은 변화를 겪어 왔다. -이후 생략 [정책제언] 대전·충남지역 지방은행의 경쟁력 강화방안 _ 김재필 Ⅰ. 머리말 1967년 부산은행과 대구은행의 설립을 시작으로 지역경제의 발전을 위한 자금공급 및 지역의 금융 중개기능 강화를 목표로 전국 각 시도에 설립된 모두 10개의 지방은행은 1970년 이래 괄목할 만한 성장을 이루어 왔다. -이후 생략 [시사칼럼]충남농정의 전개방향 _ 남궁영 Ⅰ. 추진배경 지난 94년, UR이 타결되고 WTO의 신국제경쟁사회가 열리면서 우리 농업에 대한 걱정의 소리와 우리 농민들의 앞날에 대한 불안감이 최고조에 달했다. -이후 생략 [연구원소식]연구원사업 연구원 시무식 丁丑年 한 해를 새롭게 시작하는「97년도 시무식」이 지난 1월 3일(金) 오전 10시 안승주 원장을 비롯한 전 직원이 참석한 가운데 본원 회의실에서 개최되었다. -이후 생략[특집]충남농업의 VISION과 대응전략 충남농업의 어제와 오늘 _ 이종상 21세기 한국농업의 발전전략과 미래모습 _ 조재환 개방화 시대에 있어서 충남농업의 대응전략 _ 최병익 농산물 유통구조 개선방안 _ 정태호 [정책제언] 대전·충남지역 지방은행의 경쟁력 강화방안 _ 김재필 서산기지 민항취항과 지역사회 파급효과 _ 이인배 [시사칼럼]충남농정의 전개방향 _ 남궁영 [연구원소식]연구원사업·원장동정·연구원활