중간엽 줄기세포 유래 치료용 나노베지클을 이용한 중추신경계 손상 치료

학위논문(박사)--서울대학교 대학원 :공과대학 화학생물공학부,2019. 8. 김병수.뇌졸중, 뇌출혈, 외상성 뇌 손상, 그리고 척수 손상과 같은 중추신경계 손상 질환은 장기적인 신체 장애, 더 나아가 죽음까지 이르게 하는 원인이다. 새로운 조직 재생 치료 전략으로서, 중간엽줄기세포 유래 엑소좀을 이용한 중추신경계 손상 치료는 최근 각광을 받고 있다. 중간엽줄기세포 유래 엑소좀은 그 만의 독특한 성질 때문에 중추 신경계 손상을 비롯한 다양한 질병에서 활발히 연구되어 왔다. 그러나 중추신경계 손상 치료를 위해 엑소좀을 사용하는 방식에는 여전히 한계가 남아 있다. 극소량의 엑소좀만이 중간엽줄기세포에서 분비되며, 엑소좀을 전신 주사 (정맥 주사) 하여도 표적 조직에 잘 도달하지 않는다. 따라서 본 주제에서는 산화철 나노입자를 중간엽줄기세포에 처리하였고 해당 세포로부터 엑소좀-모방 나노베지클을 분리하였다. 산화철 나노입자는 중간엽 줄기세포 내에서 철 이온으로 이온화 되며, 해당 이온들은 세포의 JNK/c-JUN 세포 신호전달 체계를 자극하여 다양한 성장 인자의 발현을 촉진시킨다. 따라서 해당 세포로부터 분리한 나노베지클은 자성을 띄는 산화철 나노입자 뿐만 아니라 고용량의 성장 인자가 탑재되어 있다. 본 연구에서는, 산화철 나노입자가 탑재된 나노베지클을 중추 신경계 병변으로 체내 전달하였고 그에 따른 치료효과를 제시한다. 먼저, 제 3장에서는 철나노입자가 처리된 중간엽줄기세포에서 철나노입자가 탑재된 철-나노베지클을 분리하였고, 이를 마우스 척수손상 모델에 전신 주사하여 외부 자장의 도움으로 손상된 척수에 전달되었다. 철나노입자가 처리된 중간엽줄기세포는 세포 자체의 큰 직경 때문에 대부분 폐에 걸렸지만, 나노미터 크기의 철-나노베지클은 폐 모세혈관을 통과하여 자기장이 있는 상태에서 손상된 척수에 상당 부분 축적되었다. 척수 병변에 축적 된 철-나노베시클은 혈관 재건에 기여하였고, 염증성 대식세포를 항염증성 아형으로 분화시켰으며, 성상교세포증을 감쇠하고 신경 세포 사멸을 억제 하였다. 결과적으로 동물 행동 검사에 의해 평가 된 바와 같이, 손상된 척수에 축적된 철-나노베지클은 치료 효과를 발휘하였고 척수 기능을 개선시켰다. 제 4 장에서는 중간엽줄기세포에서 유래하였고 철나노입자가 함유 된 자성 철-나노베지클을 랫드 허혈성 뇌졸중 모델에 정맥 주사 하였다. 동물 모델은 과도 중간 대뇌 동맥 폐색 수술을 통해 허혈성 뇌졸중을 유발했다. 세포 실험을 통해, 철-나노베지클은 대조군 나노베지클과 비교하여 다양한 유형의 세포에서 증진된 혈관 생성 효과, 세포 사멸 억제 효과, 그리고 항염증 효과를 나타내었다. 동물 실험에서는, 철-나노베지클의 정맥 주사 직후에 외부 자기장으로 작용하는 자석 헬멧을 동물 모델에 씌웠다. 그리고 동물 형광 촬영을 통해 좌뇌 반구 (뇌졸중 병변)에 철-나노베지클이 축적됨을 관찰하였다. 주사 후 3 일째에 경색 부위 크기를 정량화 한 결과, 외부 자기장 존재 아래 철-나노베지클을 투여 한 실험군에서 경색 부위가 대조군에 비하여 현저하게 감소되었다. 또한, 동물 행동 실험을 통하여 철-나노베지클 (자기장 O) 의 주입 및 치료가 동물 모델의 운동 기능을 향상 시킨다는 것을 입증하였다. 따라서 본 연구에서는 산화철 나노입자가 처리된 중간엽줄기세포로부터 분리한 나노베지클은 고용량의 성장 인자뿐만 자석 항법 도구로 쓰일 수 있는 산화철 나노입자를 함유하며, 외부 자기장을 통해 손상된 척수 또는 대뇌 병변에 표적 전달되는 것을 확인했다. 이러한 철-나노베지클은 중추신경계 손상 치료를 위한 우수한 치료제로서 활용될 수 있으며, 통상적인 중간엽줄기세포 기반 또는 중간엽줄기세포 유래 엑소좀 기반의 치료 기법을 대체할 수 있다. 본 연구의 기술은 미래에 중추신경계 손상의 성공적인 치료를 위해 사용 될 수 있을 것이다.Central nervous system (CNS) injuries such as ischemic stroke, hemorrhage, traumatic brain injury, and spinal cord injury (SCI) are leading cause of long-term disability or death. As a novel tissue regenerative strategy, application of mesenchymal stem cell-derived exosomes (MSC-exosomes) for treatment of CNS injuries has drawn much attention. Owing to their unique properties, MSC-exosomes have been actively studied for treatment of wide variety of diseases including CNS injuries. However, it still remains challenging to improve the therapeutic outcomes of using exosomes to treat CNS injuries. Very small quantities of exosomes are released from MSC, and MSC-exosomes poorly accumulate in target tissue after systemic administration. Thus, iron oxide nanoparticles (IONP) were introduced to MSC and exosome-mimetic nanovesicles were isolated from those IONP-treated MSC. Treatment of IONP contribute to enhanced expression of therapeutic growth factors in MSC, which are attributed to IONP that are slowly ionized to iron ions which activate the JNK and c-Jun signaling cascades in MSC. Isolated nanovesicles incorporate not only IONP which act as magnet-navigating tool, but also large amount of therapeutic growth factors. In this thesis, in vivo systemic delivery of IONP-harboring nanovesicles and their therapeutic effects in CNS lesions are presented. First, IONP-haboring nanovesicles (NV-IONP) were fabricated by serial extrusion of IONP-treated MSC (MSC-IONP), and systemically delivered to injured spinal cord in mouse SCI model with help of external magnetic field (MF). While intravenous injection of MSC-IONP resulted in severe lung accumulation due to their large size, nano-sized NV-IONP evaded lung entrapment and significantly accumulated in injured spinal cord in presence of MF. NV-IONP accumulated in SCI lesion contributed to blood vessel reconstruction, polarized inflammatory M1 macrophages to anti-inflammatory M2 subtype, attenuated astrogliosis, inhibited neuronal death. Consequently, increased amount of NV-IONP accumulated in injured spinal cord exerted therapeutic effects and improved the spinal cord function, as evaluated by Basso Mouse Scale (BMS) behavioral test. Second, IONP-incorporated magnetic nanovesicles (MNV) derived from MSC were intravenously administered to rat ischemic stroke models. Animals received middle cerebral artery occlusion (MCAO) to induce ischemic stroke. In vitro, MNV exerted enhanced angiogenic, anti-apoptotic, and anti-inflammatory effect in various types of cells, as compared to control nanovesicles (NV). In vivo, we applied magnet helmet which act as an external MF, to rat MCAO models immediately after intravenous injection of MNV. Fluorescence imaging showed successful accumulation of MNV specifically in left hemisphere of brain (ischemic lesion). Quantification of infarcted area at 3 days after treatment revealed that administration of MNV in presence of MF resulted in significant reduction of infarcted area compared to control group. Limb placement test (LPT) also demonstrated that treatment of MNV (MF+) improves the functional behavior of MCAO-received animals. Thus, we observed that the nanovesicles synthesized from IONP-laden MSC contain both greater amounts of reparative growth factors and IONP which act as a magnet-guided navigation tool toward injured spinal cord or cerebral lesion. These IONP-harboring nanovesicles derived from MSC can serve as excellent therapeutic agent for treatment of CNS injuries, and can be a preferable replacement of conventional MSC or MSC-exosome therapy. This technology can be used in the future for successful therapy of CNS injuries.Chapter 1. Research background and objectives 1 1.1. Challenges and limitations of MSC-based therapeutics 3 1.2. Extracellular vesicles 5 1.3. Therapeutic potency of MSC-derived EVs. 8 1.3.1. In vitro effects on target cells 8 1.3.2. Pre-clinical studies in various disease models 9 1.4. Exosome-mimetic nanovesicles 10 1.5. Magnetic drug delivery 12 1.6. Research objectives of the thesis 13 Chapter 2. Experimental procedures 15 2.1. Cell culture 17 2.2. Preparation and characterization of materials 18 2.2.1. IONP synthesis 18 2.2.2. Fabrication of NV-IONP derived from hMSC-IONP 19 2.2.3. Characterziation of NV-IONP 20 2.3. In vitro assays 21 2.3.1. IONP uptake by hMSC 21 2.3.2. Angiogenic effect of NV-IONP 22 2.3.3. Evaluation of anti-apoptosis in neuronal cells. 23 2.3.4. Assessment of astrocyte modulation 24 2.3.5. Macrophage polarization 25 2.3.5.1 Preparation of rBMDM 25 2.3.5.2 Evaluation of macrophage phenotype 26 2.3.6. qRT-PCR, western Blot, ELISA, and protein array assay 27 2.4. In vivo studies 28 2.4.1. Experimental animal models 28 2.4.1.1. SCI models 28 2.4.1.2. MCAO ischemia-reperfusion models 29 2.4.2. In vivo biodistribution 30 2.4.2.1. Biodistribution of MSC-IONP and NV-IONP in SCI mouse model 30 2.4.2.2. MNV biodistribution in MCAO rat model 31 2.4.3. Animal group assignment 32 2.4.3.1. SCI mouse model 32 2.4.3.2. MCAO rat model 33 2.4.4. Therapeutic mechanism of NV-IONP 34 2.4.4.1. Evaluation of apoptosis and fibrotic scar formation in SCI model 34 2.4.4.2. Immunohistochemistry and western blot analysis of extracted spinal cord 35 2.4.4.3. Western blot and IHC analysis of extracted brain tissue of MCAO model 36 2.4.5. Evaluation of therapeutic effects from accumulated NV-IONP 37 2.4.5.1. Hindlimb locomotor score of SCI model 37 2.4.5.2. Measurement of cerebral infarct volume of MCAO model 38 2.4.5.3. Functional behavior test of MCAO model 39 2.5. Statistical analysis 40 Chapter 3. Therapeutic efficacy-potentiated and diseased organ-targeting nanovesicles derived from mesenchymal stem cells for spinal cord injury treatment 41 3.1. Introduction 43 3.1. Result and discussion 47 3.2.1. IONP internalization into hMSCs and up-regulation of growth factors 47 3.2.2. Characterization of nanovesicles isolated from hMSC-IONP 51 3.2.3. Enhanced angiogenesis and anti-apoptotic effect of NV-IONP 60 3.2.4. Stimulation of therapeutic growth factor expression in astrocytes by NV-IONP 64 3.2.5. In Vitro attenuation of inflammatory responses and M2 polarization in macrophages 65 3.2.6. Spinal cord targeting of NV-IONP via external magnetic guidance 70 3.2.7. Enhanced angiogenesis and M2 polarization in injured spinal cord by magnet-guided NV-IONP 74 3.2.8. Attenuation of glial scar formation and enhanced functional recovery by magnet-guided NV-IONP 79 Chapter 4. Stem Cell-derived Magnetic Nanovesicles Target and Attenuate Ischemic Stroke 81 4.1. Introduction 83 4.2. Result and discussion 86 4.2.1. IONP uptake and enhanced therapeutic growth factors in MSC 86 4.2.2. IONP and higher level of therapeutic growth factors in MNV 92 4.2.3. In vitro therapeutic effects of MNV 96 4.2.4. Magnet-assisted ischemic lesion targeting of MNV 100 4.2.5. In vivo therapeutic effect and mechanism of MNV for treatment of ischemic stroke 105 Chapter 5. Conclusion 107 References 111 요약 (국문 초록) 132Docto

트리아진으로 가교된 표피형 고분자 지지체의 개발과 고체상 합성에의 응용

Thesis(doctoral)--서울대학교 대학원 :응용화학부,2005.Docto

셀프서비스에서의 대기 행렬 : 호혜성과 사회적 압력

학위논문(석사) - 한국과학기술원 : 경영공학부, 2016.2 ,[v, 38 p. :]기술이 발전해감에 따라 많은 서비스들이 셀프서비스로 전환되고 있다. 일반적으로 셀프서비스라 함은 기술을 이용한 기술 기반의 셀프서비스의 형태를, 예를 들어 ATM 기계나 셀프 계산대, 떠올리기 쉬우나 최근 마케팅의 일환으로 사용되고 있는 체험서비스 역시 셀프서비스라 할 수 있다. 대표적으로 애플의 애플스토어에서는 고객들이 제품을 직접 만져보고 사용해보며 체험이라는 서비스를 스스로 얻어간다. 즉 체험이라는 셀프서비스가 제공되는 것이다. 2015년 애플워치가 출시되면서 애플은 고객들에게 그를 체험할 수 있는 시간에 15분이라는 제약을 걸었다. 그렇지 않으면 체험 서비스에 대한 수요를 다 만족시킬 수 없기 때문이다. 우리는 이와 같은 고민에서 출발하여, 고객이 서비스 시간을 결정하는 셀프서비스의 생산성을 향상시키기 위한 연구를 진행하였다. 서비스 조직에서는 고객이 부분적 직원으로 활동하며 서비스의 생산 과정에 참여한다. 특히나 셀프서비스에서는 고객이 유일한 서비스의 생산자이자 소비자가 된다. 우리는 고객의 서비스 시간 결정에 영향을 미치는 많은 요소들 중 행동학적 요소로 호혜성과 사회적 압력을 지목했다. 호혜성은 도움 받은 것에 대해서 보답하고 해를 입은 것에 대해서는 보복하는 사회적 선호 중의 하나이다. 셀프서비스에는 앞사람이 사용한 시간만큼 내가 기다려야 하며, 내가 사용하는 만큼 뒷사람을 기다리게 하므로, 대기 시간은 이러한 호혜성의 대상이 될 수 있다고 예상했다. 또한 사람들은 사회적인 압력이 있을 때 좀 더 친-사회적 결정을 내리는 성향이 있으며 이러한 사회적 압력은 실제 사람들이 아닌 눈 모양의 그림과 같은 조그마한 신호로도 줄 수 있다는 것이 잘 알려져 있다. 셀프서비스에서 뒷사람을 위해 짧게 사용하는 것은 친-사회적 행동이므로 많은 사람들이 기다리고 있는 것은 사회적 압력으로 작용할 때 사람들이 더 짧게 사용할 것이라 예상했다. 본 연구는 퀄트릭스를 사용해 작성한 시나리오 기반의 설문지를 아마존의 엠턱 서비스를 통해 전세계인을 실험대상으로 온라인 설문조사를 하였다. 대기시간 (최대 9분 중 3, 6, 9분) *사회적 압력(유, 무)의 총 6가지 시나리오를 제작하여 가설을 확인하고자 했다. 온라인 설문 조사이므로 그림을 이용하고 클릭이라는 행동을 요구함으로써 최대한 현실감을 높이려고 하였다. 결과적으로 사람들은 앞사람이 많이 혹은 적게 기다리게 하면, 그 또한 뒷사람을 많이 혹은 적게 기다리게 하는 호혜적인 행동을 보이는 것을 확인 했다. 사회적 압력은 전체 서비스 시간을 줄여주진 못했지만, 앞사람이 많이 사용하는 경우에 나타날 수 있는 부정적인 형태의 호혜성을 억제하는 것으로 나타났다. 추가적인 실험을 통해 더 구체적인 맥락의 시나리오를 제공하면 사회적 압력의 효과가 더 커지는 것을 확인하였다. 본 연구는 호혜성이 금전적인 대상이 아닌 대기 시간의 개념에도 적용되는 지를 확인했다는 것에 큰 의미가 있다. 또한 단순 설문조사가 아닌 실험을 엠턱을 통해 수행했고, 그를 위해 실험 디자인에 많은 노력을 기울였고 클릭이라는 독창적인 실험 기법을 사용했다는 점에 큰 의의를 둔다. 앞으로 셀프서비스가 더욱 확장됨에 따라 대기 행렬이나 서비스 생산성에 대한 고민이 늘어날 것인데, 본 연구가 그에 대한 개선 아이디어를 제공 할 수 있을 것이다.한국과학기술원 :경영공학부

신재생에너지 복합전력시스템 최적화 : 제철소 사례연구

학위논문(석사) - 한국과학기술원 : 기술경영전문대학원, 2016.8 ,[iii, 45 p. :]It has become increasingly important to eliminate fossil fuel as a power generation source. Fossil fuel is one of the main sources of greenhouse gases, and the Korean government has officially announced its aggressive plan for reducing greenhouse gases. As a result, the Korean government has been expanding its renewable energy generation facilitiestherefore, the reduction of fossil fuel is a major policy issue. In this context, expanding renewable energy generation facilities can be a viable measure for reducing the use of fossil fuel in urban areas. This study evaluated the feasibility of a renewable energy hybrid system in Gwangyang-si, which has the lowest degree of carbon emissions among the Korean municipalities. To facilitate this analysis, HOMER(Hybrid Optimization for Electric Renewables) was used to determine the following factors: power grid features, actual power consumption, renewable and sustainable energy sources and distribution facilities. HOMER is the economic feasibility analysis simulation program for renewable energy hybrid systems that was developed by NREL(National Renewable Energy Laboratory). HOMER is an optimization program that aims to find a small-scale power configuration combination that minimizes cost by designing off-grid and grid-connected power systems. This software analyzes a variety of renewable energy sources. In this study, a sensitivity analysis was conducted on the renewable energy hybrid system that was built based on the monthly mean wind velocity and the fuel price of Gwangyang-si for 2014. This sensitivity analysis found that the PV 5%,grid 95% hybrid system was better for the environment than the grid-diesel system. If a carbon tax is introduced, the PV 5%, grid 95% hybrid system has a higher. The levelized COE(cost of electricity) of the grid-diesel system was compared with that of the PV-grid hybrid system. The results showed that the COE of the PV-grid system was higher than that of the grid-diesel system. However, the PV-grid system was much better for the environment because the quantity of greenhouse gases emitted was substantially decreased. The annual $CO_2$ emissions of the grid-diesel system were 752,921kg/yr, whereas the annual $CO_2$ emissions of the PV-grid hybrid system were 722,739kg/yr. Thus, the PV-grid hybrid system can substantially reduce the discharge fee over 30 years. Moreover, if there is a penalty for discharging carbon, then the difference between the NPC(net present cost) for 5% renewable energy and the NPC of the current system would decrease. That is, there would be a higher ROI(return on investment) for renewable energy if a carbon tax was levied. Recently, the environmental pollutant discharge fee has been increasing. For instance, the EU is planning to increase the environmental pollutant discharge fee. Thus, the investment value of renewable energy will likely increase. The technical feasibility of a renewable energy hybrid system that is modeled based on an energy storage facility such as a fuel battery is also analyzed. The limitations of this paper and possible future studies will be discussed in the last section of this paper.한국과학기술원 :기술경영전문대학원

Focused Update on Aspirin for Primary Stroke Prevention in Korean Clinical Practice Guidelines for Stroke

The first edition of the Korean clinical practice guidelines for primary stroke prevention reflects evidence published before June 2007. Since then, several clinical studies and meta-analyses have been conducted to determine the efficacy of aspirin for the primary prevention of cardiovascular disease including stroke. The aim of this guideline update is to provide timely recommendations taking into consideration the new evidence. Three clinical studies and four meta-analyses performed between July 2007 and November 2010 were identified and included for updating the guidelines. The main finding was a lack of aspirin efficacy for primary stroke prevention in patients with diabetes or peripheral arterial disease. We have summarized the new evidence and revised our recommendations for aspirin for primary stroke prevention. New evidence will need to be reflected continuously in future guideline updates.N

2011 Update of Scientific Statement for the Primary Prevention of Stroke: Dyslipidemia and Inflammation

Background: This scientific statement is intended to provide a systematic review of new evidences in dyslipidemia and inflammation for primary stroke prevention. Methods: Using a structured literature search, we identified major observational studies, clinical trials, meta-analyses,and updated major guidelines published between July 2007 and November 2010. In addition to the brief summary of earlier evidences employed in the first edition of Korean clinical practice guideline for primary prevention of stroke, we summarized the newly identified evidences. Results: For dyslipidemia, observational studies further support a strong association between ischemic stroke and high total and low-density lipoprotein cholesterol and low high-density lipoprotein cholesterol. Two clinical trials and 6meta-analyses confirm statin efficacy for primary prevention of stroke in high risk patients. Efficacy of other lipid-lowering agents is not established. For inflammation, inflammatory markers might help to identify patients having high risk for stroke or cardiovascular event and to decide whether statin therapy is indicated, but its usefulness for broad population needs to be confirmed. Conclusions: Writing committee will continue to keep an eye on upcoming evidences to timely update the guideline for primary stroke prevention in dyslipidemia and inflammation.N

Management of Asymptomatic Carotid Stenosis for Primary Stroke Prevention: 2012 Focused Update of Korean Clinical Practice Guidelines for Stroke

Extracranial carotid stenosis is a well-established, modifiable risk factor for stroke. Asymptomatic extracranial carotid stenosis is increasingly being detected due to the introduction of less-invasive and more-sensitive advanced diagnostic technologies. For severe asymptomatic stenosis, earlier pivotal clinical trials demonstrated the benefit of carotid endarterectomy over the best medical therapy. Since then, great advances have been made in interventional and medical therapies as well as surgical techniques. The first edition of the Korean Stroke Clinical Practice Guidelines for primary stroke prevention for the management of asymptomatic carotid stenosis reflected evidences published before June 2007. After the publication of the first edition, several major clinical trials and observational studies have been published, and major guidelines updated their recommendation. Accordingly, the writing group of Korean Stroke Clinical Practice Guidelines (CPG) decided to provide timely updated evidence-based recommendations. The Korean Stroke CPG writing committee has searched and reviewed literatures related to the management of asymptomatic carotid stenosis including published guidelines, meta-analyses, randomized clinical trials, and nonrandomized studies published between June 2007 and Feb 2011. We summarized the new evidences and revised our recommendations. Key changes in the updated guidelines are the benefit of intensive medical therapy and further evidence of carotid artery stenting as an alternative to carotid endarterectomy. The current updated guidelines underwent extensive peer review by experts from the Korean Stroke Society, Korean Society of Intravascular Neurosurgery, Korean Society of Interventional Neuroradiology, Korean Society of Cerebrovascular Surgery, and Korean Neurological Association. New evidences will be continuously reflected in future updated guidelines.N