Lymph node-targeting and antigen-presenting proteinticles for in vivo cancer immunotherapy

학위논문 (석사)-- 서울대학교 융합과학기술대학원 : 분자의학 및 바이오제약학과, 2015. 8. 이동수.Cancer immunotherapy is a new class of cancer treatment which uses the bodys own immune system to defeat cancers. To elicit immune responses that attack cancer cells, efficient antigen delivery to lymph nodes (LNs) can be an appropriate strategy, because LNs possess a high population of immune-related cells such as antigen presenting cells (APCs), T cells and B cells. However, realization of this strategy remains challenging. Biocompatible protein-based nanoparticles (named proteinticles) that self-assemble inside cells with precise 3D nanostructure, have a unique advantage over synthetic nanomaterials for LN-targeting immunotherapy. Herein, human ferritin heavy chain (HFt) and red fluorescence protein (RFP) were selected as a promising antigen carrier and a model antigen, respectively. Genetically recombined HFt-RFP vaccine particle with a size of 26 nm showed fast diffusion and long retention in the targeted lymph node. Moreover, HFt-RFP induced strong cytotoxic CD8+ T cell responses, resulting in significant inhibition of tumor growth in RFP-expressing tumor-bearing mice. Here, we show that LN-targeting strategy using antigen-presenting proteinticles holds promises for effective cancer immunotherapy.ABSTRACT ……………………………………………………… i Table of Contents ……………………………………………… ii List of Tables …………………………………………………… iv List of Figures ……………………………………………………v 1. Introduction ……………………………………………………1 2. Materials and Methods 2.1 Biosynthesis of HFt-RFP ……………………………… 3 2.2 Characterization of HFt-RFP ……………………………4 2.3 Preparation of Cy5.5 …………………………………… 4 2.4 Lymph node accumulation …………………………… 5 2.5 Intracellular cytokine staining ………………………… 5 2.6 Evaluation of tumor inhibition effect ………………… 6 2.7 Immunofluorescence dual-staining and lymphocytes population analysis …………………………………… 7 2.8 Statistical analysis ………………………………………8 3. Results 3.1 Development and characterization of HFt-RFP …… 9 3.2 Comparison of HFt-RFP with RFP, HFt in time- dependent in vivo and ex vivo biodistribution …… 14 3.3 Real-time tracking of HFt-RFP to the regional lymph node …………………………………………………… 19 3.4 Model tumor antigen-displaying proteinticles as an immunotherapeutic vaccine ………………………… 21 3.5 In vivo antitumor effect ……………………………… 24 3.6 Histological analysis ………………………………… 27 4. Discussion ………………………………………………… 29 5. Conclusion ………………………………………………… 31 6. References ………………………………………………… 32Maste

(The) effect of topical citric acid application on the healing of experimental intrabony pocket

치의학과/석사[한글] 치주질환으로 파괴된 치주조직의 치유에 국소적 구연산도포가 미치는 영향에 관하여 알 아보기 위하여 산처리유무에 따른 치주조직의 치유과정을 조직병리학적으로 관찰하였다. 성견 제 3, 4 소구치의 근심치근에 치조골하방 3mm 깊이의 3 wall intrabony pocket을 외과적으로 형성하고 9주가 지난뒤, 치료를 하지않은 대조군과 치근활택술 및 치은판막술 을 시행한 실험군, 치근활택술과 치은판막술 및 pH 1 구연산 처리를 함께 한 실험군으로 나누어 실험후 2, 4, 8, 16주째 실험동물을 각각 2마리씩 희생시켜 Hematoxylin과 Eosin 염색 및 Gomori's trichrome 염색을 하여 광학현미경으로 관찰한 결과 다음과 같은 소견 을 얻었다. 1. 상피세포의 근단이동은 대조군에서는 실험기간동안 지속되었으나 실험군에서는 8주 이후부터 근단이동을 볼 수 없었고, 실험군간의 차이점은 없었다. 2. Supracrestal fiber의 부착은 대조군에서는 4주까지 전혀 관찰되지 않았으나 두 실 험군에서는 산처리에 관계없이 4주부터 관찰되었다. 3. 두 실험군에서 산처리에 관계없이 백아질재생이 2주부터 관찰되었으며 대조군에서는 전 실험기간을 통하여 관찰되지 않았다. 4. 골내낭기저부에서의 치조골형성은 대조군은 16주에서, 두 실험군에서는 2주부터 다 양하게 관찰되었다. 5. 치주인대의 재생은 대조군에서는 실험 전기간에 걸쳐 관찰되지 않았으나 실험군에서 는 산처리유무에 관계없이 8주부터 부분적으로 결함조직섬유의 재부착이 관찰되었고, 16 주에는 치주인대와 같은 기능적 배열을 보였다. 6. 결합조직내 염증세포침윤은 실험기간이 경과함에 따라 감소되었으며, 대조군보다 실 험군에서 미약하게 관찰되었다. [영문] This study was undertaken to observe histopathologic changes of periodontal tissues in dogs after surgical periodontal thereapy with or without acid treatment, and to evaluate the effect of topical application of citric acid on the pathologically exposed root surfaces. In this experiment,8 dogs were used and divided into untreated control, surgical experimental and acid treated experimental groups. After 9 wks of making intrabony pocket, gingival flap was reopened for surgical and acid demineralizing treatment. Thereafter, the dogs were serially sacrificed each 2 dogs every 2,4,8 and 16 wks. after surgical or acid treatment. The specimens were obtained in block and made the original slides cut with a thickness of 8μm, and stained with hematoxylin and eosin, and Gomori's one-step trichrome method. The results were as follows; 1. The continuous apical migration of epithelium was observed in untreated control group, however there was no apical migration in two experimental groups from 8 wks. 2. The supracrestal fiber attachment was lost until 4 weks., in control group. But it was regained from 4 wks. in in 2 experimental groups regardless of acid treatment. 3. Throughout the experimental periods, new cementum was observed in 2 experimental groups except control. 4. There was various bone regeneration in the intrabony notch area at 16 wks. of untreated control and all experimental periods of 2 experimental groups. 5. In the all experimental groups, ligamental fiber adhesion was observed until 4 wks. and new attachment appeared from 8 wks. in the inferior notch area. But there was not ligament regeneration in the control group. 6. The infiltration of inflammatory cells decreased according to advance of experiment, and was observed lesser in experimental groups than untreated control.restrictio

(The) effects of oxidized cellulose membrane in conjuction with replamineform hydroxyapatite or resorbable calcium carbonate on the periodontal he

치의학과/박사[한글] 치주질환치료의 궁극적인 목적은 손상된 치주조직의 재생과 기능회복에 있다. 치조골결 손부를 회복하기 위해서는 합성골이식이 사용될 수 있으며 치은상피와 결합조직에서 유래 하는 세포들의 근단이동을 효과적으로 억제시키기 위하여는 치주조직유도재생술이 사용되 고 있다. 본 연구의 목적은 porous replamineform hydroxyapatite(HA)와 porous resorbable calc ium carbonate를 치조골결손부에 이식한 후 흡수성 oxidized cellulose membrane을 혼합 사용한 경우 치주조직의 재생과 치유에 미치는 영향을 알아보기 위함이다. 이를 위해 성견 소구치와 대구치에 교정용 탄성사를 결찰하여 만성치주염을 유발시킨후 치은박리수술만 시행한 경우를 대조군으로, 치은박리수술시 membrane을 사용한 경우를 실험Ⅰ군으로, 수술시 porous HA를 이식하고 membrane을 사용한 군을 실험Ⅱ군, 수술시 c alcium carbonate를 이식하고 membrane을 사용한 군을 실험Ⅲ군으로 설정하였다. 수술후 각 1, 3, 6, 12, 26주의 치유결과를 조직학적으로 비교 관찰하여 다음과 같은 결과를 얻 었다. 1. Oxidized cellulose membrane은 1주에 대부분이 흡수된 소견을 보였으며, 3주 이후 에는 관찰되지 않았다. 2. 접합상피는 대조군에서 1주에 notch 부위까지 이동을 보이고 26주까지 지속되었으며 , 실험Ⅰ군은 1주에, 실험Ⅱ, Ⅲ군은 3주에 notch 상부에 이르렀으나 모든 실험군에서 전 실험기간동안 notch 하부로의 근단이동은 관찰되지 않았다. 3. 결합조직내의 염증세포침윤은 1주에서 대조군과 실험Ⅰ군에서 실험Ⅱ, Ⅲ군에 비하 여 심하게 나타났으며 3주부터 모든군에서 감소하여 6주이후에는 차이점이 나타나지 않았 다. 4. 신생백악질형성은 대조군에서는 전실험기간동안 관찰되지 않았으며 실험Ⅰ, Ⅱ, Ⅲ 군에서는 6주부터 관찰되어 26주에는 모든 실험군의 notch 상방까지 형성되는 소견을 보 였다. 5. 결합조직섬유의 치근과의 배열양상은 대조군에서는 치근과의 부착이 나타나지 않았 으며 실험 Ⅰ, Ⅱ, Ⅲ군에서는 6주까지 치근과 나란히 주행하고 12주부터 일부 수직배열 되어 26주에는 치밀해지는 소견을 보였다. 6. 신생골은 대조군에서는 전실험기간동안 관찰되지 않았으며, 실험 Ⅰ군의 6주에서 no tch 부위의 골형성이 나타나고, 실험 Ⅱ, Ⅲ군에서는 3주부터 치조골과 인접한 이식재료 편 주변에 생성되기 시작하여 26주에는 notch 상방까지 형성되는 소견을 보였다. 이상의 결과로 볼때 oxidized cellulose membrane은 접합상피의 근단이동과 신생백악질 형성에는 효과를 보였으나 치조골 형성 능력은 미약한 것으로 보이며, porous HA와 calc ium carbonate 이식재는 치주조직의 치유와 재생에 좋은 결과를 나타내고 재료간의 차이 점은 적은 것으로 사료된다. [영문] The ultimate goal of periodontal therapy is to fully reconstruct the dental attachment apparatus. Commonly used techniques for treatment of intrabony defects include a combination of root planing, currettage and grafting. To prevent the apical migration of epithelial cells, the technique of guided tissue regeneration is used. The aim of this study is to evaluate the effects of oxidized cellulose membrane in conjuction with porous replamineform hydroxyapatite or porous resorbable calcium carbonate in the periodontal therapy of dogs. Experimental periodontitis was induced by the ligation of orthodontic elastic threads in the premolars and 1st molar of 5 adult dogs for 10 weeks. 4 types of regenerative procedures were performed as follows; 1) flap operation(control group) 2) flap operation with oxidized cellulose membrane covering(experimental group Ⅰ) 3) flap operation with porous replamineform hydroxyapatite grafting and oxidised cellulose membrane covering(experimental group Ⅱ) 4) flap operation with porous resorbable calcium carbonate grafting and oxidized cellulose membrane covering(experimental group Ⅲ) Thereafter, dogs were serially sacrificed at the 1, 3, 6, 12, and 26 weeks, and the specimens were prepared, and stained with hematoxylin-eosin stain for the light microscopic evaluation. The results of this study were as follows; 1. The most of oxidized cellulose membrane was resorbed at 1 week, and did not obserbed after 3 weeks. 2. Junctional epithelium(JE) of the control group migrated to the notch at 1 week and continued through the experimental period. In the experimental group Ⅰ, JE migrated toward the notch at 1 week, and in the experimental group Ⅱ and Ⅲ, at 3 weeks. But in all experimental groups JE did net reach to the notch through the experimental period. 3. In the aspects of the inflammatory cell infiltration, control and experimental group Ⅰ showed severe aggregation than experimental group Ⅱ and Ⅲ at 1 week. But it decreased from 3 weeks and there were no differences among all groups from 6 weeks. 4. New cementum was not formed in control group, but observed from 12 weeks in the experimental group Ⅰ, and 6 weeks in the experimental group Ⅱ and Ⅲ. And it grew over the notch at 26 weeks. 5. In the control group, long junctional epithelium covered the treated root, to the notch but in the experimental groups periodontal tissue was healed with new periodontal ligament from 6 weeks and become close to 26 weeks. 6. New bone was not found in the control group, but in the experimental group Ⅰ it was found at 12 weeks. In the experimental group Ⅱ and Ⅲ, new bone was formed around the implanted material near the alveolar bone from 3 weeks and grew over the reference notch at 26 weeks.restrictio