바이오프린팅과 그래핀 양자점을 이용한 효율적인 간세포 분화 연구

학위논문(박사) -- 서울대학교대학원 : 수의과대학 수의학과, 2022. 8. 강경선.미국에서는 15분마다 장기 이식 대기자 명단에 이름이 하나씩 추가된다. 장기이식에 대한 수요는 빠르게 증가하지만 대기자 명단 중 기증자로부터 일치하는 장기를 받을 수 있는 환자는 3분의 1도 되지 않는다. 이러한 부족을 완화하기 위한 가장 유망한 기술 중 하나는 세포, 공학 및 재료 방법을 결합하여 인공 장기를 생산하는 조직 공학이다. 여러 장기 중 특히 간의 경우, 약물과 독소대사를 하는 것이 가장 큰 특징이다. 또한 약물개발시 부작용이 나타나는 대표적인 장기이므로 약물개발과 독성학적 평가를 함에 있어 체외 간 모델은 필수적이다. 인공 간을 만들기 위해서는 장기를 구성할 수 있는 구조체와 많은 수의 세포가 필요하다. 장기를 구성할 수 있는 구조체는 탈세포화 과정을 거친 생체유래나 3D bioprinting 기술을 통해 ECM 성분의 구조체를 직접 프린팅 하는 방법등이 있다. 이 연구의 첫 번째 부분에서는 3D bioprinting 기술을 통해 구조체를 프린팅 한 후 스피닝 세포배양조건을 활용하여 생체내 상태와 생리적 관련이 있는 3차원 체외 간 모델을 구축하였다. 본 연구에서는 우리 몸속의 가장 큰 장기인 간을 3차원 압출기반 바이오프린팅 기술을 활용하여 프린팅하였다.. 그러나 현재 간과 같이 크기가 큰 체외 모델에서는 중앙 부위가 저산소 상태로 인해 약물과 독소의 신진대사가 느리고 약해지기 때문에 체외 간 모델에서 정확한 약물 효과를 예측하는 것은 여전히 어렵다. 여기서 우리는 육각형 바이오프린트 간 구조를 구성하고 지속적인 매체 자극이 있는 동적 조건을 통합했다. 동적 조건은 실제 간의 영양소가 풍부한 정맥과 산소가 풍부한 동맥을 포함하는 이중 혈액 공급 순환 시스템을 모사하고자 하였다. 그 결과 동적 조건 하에서 바이오프린트된 간구조의 HepG2 세포는 정적 조건의 세포에 비해 증식능력과 기능성이 향상되었다. 또한, 회전 조건에서 배양된 생체 인쇄 간 구조에서 약물 유도 신호와 반응을 촉진하는 역할을 하는 스페로이드의 수가 증가했다. 또한, 동적 조건에서 HepG2 세포는 집중적인 TGFα 유도 상피에서 중간엽으로의 전이(EMT)를 나타내었고, 아세트아미노펜 유도 간 독성에 대한 감수성을 증가시켰으며, N-아세틸시스테인(NAC)의 투여에 의한 간 독성 예방도 증가시켰다. 이중 혈액 공급 순환 시스템의 충분한 양의 산소와 영양소는 생체내 간의 높은 재생 능력을 가능하게 하는데, 우리의 연구 결과를 종합하면, 바이오 프린팅된 간 구조를 생성하는 동안 사용된 동적 조건은 부분적으로 간 손상과 복구 현상의 재현을 가능하게 한다는 것을 보여준다. 이 단순하지만 효과적인 배양 전략은 약물 효과 평가를 위한 체외 모델링을 개선하기 위해 바이오프린트된 간 구조를 용이하게 한다. 첫 연구에서는 간암세포인 HepG2를 활용한 체외 인공 간 모델을 설립하였으나 질병 모델링 및 개인 맞춤 의료에는 유도만능줄기세포가 각광 받고 있다. 그러나, 유도만능줄기세포에서 간세포로의 분화에서 낮은 분화효율은 최종적으로 많은 수의 분화된 세포를 얻기 어렵게 하기 때문에 극복해야 할 문제점 중 하나이다. 두 번째 연구에서는 커피콩 유래 그래핀 양자점을 활용하여 인간만능줄기세포에서 간세포로의 분화효율을 높여 많은 수의 간세포를 최종적으로 얻고자 하였다. 최근까지 그래핀 양자점은 다양한 세포 유형으로 줄기세포의 효율적인 분화를 촉진하는 것으로 밝혀졌다. 그러나 간 계통에서 이들의 생물학적 활성과 신호 전달 메커니즘은 완전히 조사되지 않았다. 여기서, 우리는 통합된 칼슘 이온들이 인슐린 유사 성장 인자 1 수용체(IGF1R)-AKT의 신호를 활성화하는 칼슘이 풍부한 커피콩 유래 그래핀 양자점를 활용하여 간세포 분화 속도를 촉진할 수 있다고 보고한다. 우리는 또한 커피콩 유래 그래핀 양자점 처리에서 인간 만능줄기세포에서 유래한 간 전구세포가 간세포와 유사한 세포로의 성숙을 상향 조절하고 담관세포에 대한 분화를 하향 조절하여 많은 수의 기능성 간세포를 성공적으로 얻는 경향이 있다는 것을 발견했다. 더욱이, 커피콩 유래 그래핀 양자점 처리로부터 인슐린 유사 성장인자 1 수용체의 활성화는 성장 인자에 관계없이 간세포 분화 효율을 향상시켜, 킬레이트 칼슘이 인슐린 유사 성장인자 1 수용체의 활성화에 중요한 역할을 할 수 있음을 암시한다. 이 연구 결과는 독립적인 생체 활성을 가진 커피콩 유래 그래핀 양자점이 인슐린 유사 성장 인자 1 대신 널리 사용될 수 있으며 비용 효율적인 성장 인자뿐만 아니라 잠재적 신규 분화인자로 활용될 수 있음을 시사한다.Three-dimensional (3D) bioprinting is a promising technology to establish a 3D in vitro hepatic model that holds great potential in toxicological evaluation. However, in current hepatic models, the central area suffers from hypoxic conditions, resulting in slow and weak metabolism of drugs and toxins. It remains challenging to predict accurate drug effects in current bioprinted hepatic models. In the first of this study, I constructed a hexagonal bioprinted hepatic construct and incorporated a spinning condition with continuous media stimuli. Interestingly, under spinning conditions, cells in the boprinted hepatic construct exhibited enhanced proliferation capacity and functionality compared to those under static conditions. Increased number of spheroids under spinning conditions exhibited intensive TGFβ-induced epithelial-to-mesenchymal transition (EMT) and increased susceptibility to acetaminophen (APAP)-induced hepatotoxicity as well as hepatotoxicity prevention by administration of N-acetylcysteine (NAC). These results demonstrate that the spinning condition employed during the generation of bioprinted hepatic constructs enables the recapitulation of liver injury and repair phenomena in particular. This simple but effective culture strategy facilitates bioprinted hepatic constructs to improve in vitro modeling for drug effect evaluation. In the second part, the goal of this research was to secure large amounts of human pluripotent stem cell-derived hepatcytes by increasing differentation efficiency using graphene quantum dots (GQDs). GQDs have been found to promote efficient differentiation of stem cells into a variety of cell types. However, their bioactivity and signalling mechanisms in the hepatic lineage have not been fully investigated. Here, we report that the hepatoblast differentiation rate can be promoted by using Ca2+-rich coffee bean-derived GQDs, where the incorporated calcium ions activate the signaling of insulin-like growth factor 1 receptor (IGF1R)-AKT. We also found that the human pluripotent stem cells (hPSCs)-derived hepatoblasts from the CB-GQDs treatment tended to upregulate the maturation into hepatocyte-like cells and downregulate the differentiation towards cholangiocytes, therby successfully obtaining a large number of functional hepatocyte-like cells. Moreover, the activation of IGF1R from the CB-GQD treatment enhances the hepatoblast differentiation efficiency regardless of growth factors, implying that chelated calcium may play a key role in activating IGF1R. Our findings suggest that CB-GQDs with independent bioactivity can be widely used in place of insulin-like growth factor 1 (IGF1) and represent a cost-effective growth factor as well as a potential differentiation factor. In conclusion, these findings imply that (i) dynamic condition can be a strategy to alleviate the hypoxic condition of large-sized bioprinted in vitro hepatic models to promote drug matabolism, (ii) hepatoblast differentiation of pluripotent stem cells is related to the presence of calcium, and chelated calcium in CB-GQDs help secure an affinity from IGF1R present in the phospholipid membrane through electrostatic attraction, potentially contributing to promoting hepatoblast differentation via calcium channels.1.1 INTRODUCTION 2 1.2 MATERIALS AND METHODS 5 1.2.1 Cell culture 5 1.2.2 Bioprinting of 3D Hepatic Constructs 5 1.2.3 Live/Dead Cell Staining 6 1.2.4 Quantitative RT-PCR 6 1.2.5 Histological Characterization (H&E Staining) 6 1.2.6 Immunocytochemistry 7 1.2.7 Functional Analysis 8 1.2.8 Western Blot Analysis 8 1.2.9 Drug Treatment 9 1.2.10 Statistical Analysis 9 1.3 RESULTS 13 1.3.1 Fabrication of 3D Bioprinted Human Liver Tissue 13 1.3.2 Spinning Culture Conditions Enable a Long-Term Culture Period of Bioprinted Hepatic Constructs with Consistent Hepatic Expression and Functionality of Encapsulated Cells 21 1.3.3 3D Bioprinted Hepatic Constructs Show Efficient Changes in Cellular Characteristics under Spinning Conditions 25 1.3.4 The Dynamic Microenvironment Generated by an Orbital Shaker Enables Artificial Liver Construction as a Liver Toxicity Test Model 30 1.3.5 Functional Evaluation of Bioprinted Hepatic Constructs Revealed That Hepatotoxicity Induced by APAP and Prevented by NAC Was Enhanced under Spinning Conditions 35 1.4 DISCUSSION 37박

Awareness of patients with periodontal disease under treatments at initial and maintenance phase

People are getting more interested in well-being, which gives the highest priority to health and does not sfare time and money for health. With increasing concern over and investment in healthy life, people's interest in and demand for dental health as a part of healthy life are also rising significantly and dental health is a critical element for the health of the whole body. Thus this study purposed to investigate the current state of dental health management in new patients who visited the department of periodontology of B University Hospital for the first time to get treatment for periodontal diseases and maintenance patients who had been completely cured of their symptoms and were having regular dental health management services and to suggest the importance and necessity and effectiveness of dental health management system, and for this purpose it conducted a survey using a structured self-reporting questionnaire on 190 subjects who were selected through random sampling from patients who visited the Periodontal Department of B University Hospital. Collected data were analyzed based on real numbers, percentages, χ^(2) analysis and t-test using a statistics processing program (SPSS Windows, Version 10.0). According to the results of the survey, 52.6% of the respondents were male and 47.4% were female. By age, 66.3% of the respondents aged between 40-59. As for academic qualification 39.5% were college graduates, and as for income 51.6% earned 1,000,000~3,000,000 won a month. As for the respondents' basic perception on their dental health, respondents who replied 'Healthy' was 5.1% of new patients while 48.4% of maintenance patients. As for oral management those who replied 'Healthy' were 5.1% of new patients while 48.4% of maintenance patients and as for the condition of oral health those who replied 'Have no oral malodor' were 28.9% of new patients while 66.0% of maintenance patients. Those who were using dental hospital to manage periodontal diseases were 27.9% of new patients while 97.9% of maintenance patients, and those who recognized the necessity of maintenance programs for periodontal disease patients were 88.6% of new patients while 93.6% of maintenance patients. Perception on the condition of dental health was 'less than average' in new patients while 'average' in maintenance patients, and the condition of dental health was in a positive correlation with oral malodor. Perception on oral condition management and periodontal disease management was higher in maintenance patients than in new patients. According to the results of this study as presented above, recognition of both oral condition management and periodontal disease management was higher in maintenance patients than in new patients. In addition, maintenance and management produced the effect of efficient oral health promotion and patients' perception on maintenance was also positive. Above all it is necessary to introduce and execute maintenance systems at private medical institutions in order to improve and maintain people's dental health. Moreover, it is necessary to develop systematic maintenance and management programs and to continue relevant researches to utilize maintenance systems at community dental clinics

한국전통발효음식과 전문가 치아미백을 시행한 자연치의 색조변화와의 관련성 The Relation between Korean Traditional Fermented Food and Discoloration on Bleached Tooth

Abstract This study is to examine the relation between Korean fermented food and discoloration on teeth, which have undergone profession tooth whitening. The color change of natural tooth, which has undergone professional tooth whitening, is objectively measured by dental colorimeter after being immersed in Korean fermented food for certain period. The tooth specimens used in this study are 30 pieces and measured 30 times per one tooth, 900 times totally by dental colorimeter ShadeEye NCC Ⓡ Dental Chroma Meter. As a result of analyzing the collected data by using PASW(statistical package) 18.0, the significant difference is found (p<0.001) in lightness and yellowness before and after of professional tooth whitening. The lightness and yellowness after immersion is changed (p<0.001) according to time. As a result of examining the degree of discoloration based on type of fluids, the similar difference is indicated (p=0.001, p<0.001 1,2 and 3 weeks after getting professional tooth whitening. As a result, Korean traditional fermented food influences on discoloration on bleached tooth depended on the duration of immersion and the type of fluids. However, this study failed to represent internal oral condition accurately therefore the results of this study could be altered by the internal oral condition, frequency of consumption of food, and the management oral hygiene