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Technique of Increasing the Stability of Porcine Pancreatic Islets Using Biopolymers
생체 고분자를 이용한 돼지 췌장소도세포의 안정성 증강 기술 연구

DC Field Value Language
dc.contributor.advisor변영로-
dc.contributor.author우윤주-
dc.date.accessioned2017-07-19T11:04:45Z-
dc.date.available2017-07-19T11:04:45Z-
dc.date.issued2013-08-
dc.identifier.other000000012969-
dc.identifier.urihttps://hdl.handle.net/10371/133332-
dc.description학위논문 (석사)-- 서울대학교 융합과학기술대학원 : 분자의학 및 바이오제약학과, 2013. 8. 변영로.-
dc.description.abstractPancreatic islet transplantation is considered as the most promising technique for the treatment of type 1 diabetes mellitus. However, the pancreatic islet isolation and transplantation technique have not been optimized yet because of several obstacles in vitro and in vivo works. The clinical problem in human pancreatic islet allotransplantation is the limitation of donor supply and immune rejection after transplantation. Moreover, the researchers related to the porcine islet studies suffer from the instability of porcine islet clusters-
dc.description.abstractthus, it was difficult for them to move on to in vitro experiments. The porcine islets are focused for alternative source to solve the donor limitation problem and the fragility of porcine islet clusters are fixed by re-providing a proper extracellular matrix to the cells. Here, we synthesized adequate biopolymers for islet surface modification and investigated the increase in stability of porcine islet clusters.
Rat pancreatic islets, INS-1 cells, and porcine pancreatic islets were used to determine the viability, functionality, and biopolymer coverage on cells. The cytotoxicity and material adherence on the cell surface of biopolymers were confirmed by rat islet ICCs or INS-1 cells before applied to porcine islet clusters. The relative viability of unmodified and biopolymer-grafted cells was determined by CCK-8/DNA assay or LDH cytotoxicity assay. The coverage test of FITC-labeled biopolymer-grafted cells was performed by confocal microscopy. After confirmation of biopolymer candidates, 6-arm PEG-lipid-SH and gelatin-DOPA were selected from candidate and two biopolymers were applied to the porcine islets in multi-layered system. The unmodified and biopolymer-grafted porcine islets were cultured for 7 days for morphology, functionality, viability, material coverage, and stability observations.
In result, the relative viability of unmodified and biopolymers-grafted porcine islets for 7-day culture was not shown a significant difference and also there was no significant difference in insulin functionality by dithizone staining in both unmodified and biopolymer-grafted porcine islets. The morphology of unmodified porcine islet clusters were destructed and dissociated into single cells but that of biopolymer-grafted porcine islet clusters were maintained their cluster round-shape. FITC-labeled 6-arm PEG-lipid-SH and gelatin-DOPA, were treated on porcine islets to determine the cell surface coverage of materials. Both materials were not penetrated into the cell cytoplasm but only adhere on the membrane of cells and cover the periphery of clusters. The average size of cell clusters was measured by coulter counter until 7-days of culturing and the distribution of cell size was calculated. The size of unmodified porcine cells on fifth day were decreased to half compared to the first day but the size of biomaterial-grafted groups were decreased less than one-third of the original size.
Regeneration of extracellular matrix and cell connection of porcine islets using biopolymers would be advantageous for further porcine works because the islets become easier in handling and also subsequent surface camouflage may be held based on this study. By increasing the stability and cellular interaction in islet clusters, the cellular communication and functionality of porcine islets could be increased and also the viability of the cells could be increased. Moreover, the further surface modification on porcine islets based on our suggestion would lead to inhibition of immune rejection of islets after transplantation.
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dc.description.tableofcontents1. INTRODUCTION 1
1.1 Insulin-dependent diabetes mellitus (IDDM) 1
1.2 Pancreatic islet transplantation 2
1.3 Obstacles in porcine islet isolation and transplantation 3
1.4 Cell-to-cell communications and signaling between pancreatic islet cells 6
1.5 Extracellular matrix components of pancreatic islet cells 6
1.6 Rationale 8
2. RESEARCH DESIGN AND METHODS 12
2.1 Synthesis scheme and preparation of biopolymers 12
2.1.1 Unfrafactionated heparin-N-hydroxysuccinimide 12
2.1.2 Low molecular weight heparin-deoxycholic acid 12
2.1.3 Low molecular weight heparin – lithocholic acid 13
2.1.4 Solubilized extracellular matrix 13
2.1.5 Glycol chitosan – phospholipid 14
2.1.6 Poly(acrylic acid) – phospholipid 14
2.1.7 Poly-L-lysine – phospholipid 15
2.1.8 Six-arm PEG – lipid – SH 16
2.1.9 Chitosan – catechol 17
2.1.10 Gelatin – DOPA 17
2.2 Animals 18
2.3 Pancreatic islet isolation and biopolymer grafting 18
2.3.1 Rat Pancreas islet isolation 18
2.3.2 Porcine islet isolation 19
2.4 Single-layer and multi-layer grafting system of biopolymers 19
2.5 Cell viability 20
2.6 Biopolymer coverage onto the islet cluster surface by confocal laser scanning microscopy 21
2.7 Functionality test of islet cell cluster 21
2.8 Islet cell cluster stability test using biopolymers 22
3. RESULTS 23
3.1 In vitro evaluation of synthetic biopolymers 23
3.1.1 Unfractionated heparin-NHS 23
3.1.2 Low molecular weight heparin-DOCA and Low molecular weight heparin-LCA 26
3.1.3 Solubilized extracellular matrix 30
3.1.4 Glycol chitosan-phospholipids 30
3.1.5 Poly(acrylic acid)-phospholipid 31
3.1.6 Poly-L-lysine-phospholipid 36
3.1.7 Chitosan-catechol 37
3.1.8 Multi-layered system using 6-arm PEG-lipid-SH and Gelatin-DOPA 42
4. DISCUSSION 55
5. CONCLUSION 57
6. REFERENCES 59
7. ABSTRACT (written in Korean) 65
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dc.formatapplication/pdf-
dc.format.extent50247295 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectporcine islets-
dc.subjectislet transplantation-
dc.subjectextracellular matrix-
dc.subjectcell communication-
dc.subjectporcine islet stability-
dc.subjectporcine fragility-
dc.subject.ddc610-
dc.titleTechnique of Increasing the Stability of Porcine Pancreatic Islets Using Biopolymers-
dc.title.alternative생체 고분자를 이용한 돼지 췌장소도세포의 안정성 증강 기술 연구-
dc.typeThesis-
dc.description.degreeMaster-
dc.citation.pagesviii, 67-
dc.contributor.affiliation융합과학기술대학원 분자의학 및 바이오제약학과-
dc.date.awarded2013-08-
Appears in Collections:
Graduate School of Convergence Science and Technology (융합과학기술대학원)Dept. of Molecular and Biopharmaceutical Sciences (분자의학 및 바이오제약학과)Theses (Master's Degree_분자의학 및 바이오제약학과)
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