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Induced myogenic commitment of human chondrocytes via non-viral delivery of minicircle DNA : 미니써클 유전체의 비바이러스성 전달을 통한 인간 연골세포의 근육세포로의 분화 유도
DC Field | Value | Language |
---|---|---|
dc.contributor.advisor | Nathaniel S. Hwang | - |
dc.contributor.author | 홍지은 | - |
dc.date.accessioned | 2017-07-19T05:53:22Z | - |
dc.date.available | 2017-07-19T05:53:22Z | - |
dc.date.issued | 2014-08 | - |
dc.identifier.other | 000000022252 | - |
dc.identifier.uri | https://hdl.handle.net/10371/129348 | - |
dc.description | 학위논문 (석사)-- 서울대학교 대학원 : 화학생물공학부, 2014. 8. Nathaniel S. Hwang. | - |
dc.description.abstract | Regenerative medicine covers the whole process of regenerating or replacing the human cells, tissues and organs to restore their normal function. The damaged tissues undergo wound healing process, which mostly end up with incomplete repair, and severely damaged tissues are often irreparable or difficult to be expanded. Therefore, cell therapies have widely studied to provide sufficient quantities of specifically differentiated cells. Especially, methods to change cell fate from a terminally-differentiated stage to another cell lineage or to multipotent/pluripotent state have been investigated. Though induced pluripotent stem cells (iPSCs) can be differentiated into the desired state, iPSC technology is currently time-consuming process and it has cell-type dependent reprogramming efficiency. Furthermore, clinical safety and genetic/epigenetic fidelity still remain as major concerns. In order to bypass inducing pluripotent stage, transdifferentiation/direction conversions have recently been developed. Objective of this thesis is to utilize non-viral system to deliver minicircle DNA vector (MCDNA, polycistronic vector containing reprogramming defined factors) to induce transdifferentiation from human chondrocytes into myogenic cells along with SB-431542, an inhibitor of the activin receptor-like kinase receptors. In order to increase the efficiency of gene delivery into chondrocytes, MCDNA was delivered into the chondrocytes via electroporation followed by poly (β-amino esters) (PBAE) transfection. The transfected cells were sorted by fluorescence-activated cell sorting (FACS), cultured on a Matrigel coated plate, passed onto multi-well plate, and then treated by SB-431542 to induce the myogenic commitment. This study demonstrated that human chondrocytes can be induced to be efficiently transdifferentiated into myogenic cells through the non-viral system. | - |
dc.description.tableofcontents | Abstract.............................................................................................................................1
Contents............................................................................................................................3 List of Figures and Table...................................................................................................5 Chapter 1. General Introduction........................................................................................6 1.1 Duchenne muscular dystrophy..........................................................................6 1.2 Non-viral delivery system and transfection......................................................7 1.3 Transdifferentiation and reprogramming..........................................................8 Chapter 2. Preparation of minicircle DNA and Polymeric Gene Carrier..........................9 2.1 Introduction.......................................................................................................9 2.2 Methods...........................................................................................................10 2.2.1 Synthesis of PBAE...................................................................................10 2.2.2 Minicircle DNA preparation....................................................................10 2.2.3 Agarose gel electrophoresis.....................................................................11 2.2.4 Dynamic light scattering analysis of MCDNA/PBAE.............................11 2.2.5 Cell culture...............................................................................................12 2.2.6 FACS analysis of transfected chondrocytes.............................................13 2.3 Results.............................................................................................................14 2.3.1 Preparation of MCDNA...........................................................................14 2.3.2 Optimization of MCDNA/PBAE weight ratio.........................................14 2.3.3 Properties of MCDNA/PBAE nanoparticles...........................................14 2.3.4 Transfection efficiencies of electroporation and MCDNA/PBAE system......................................................................................................15 2.4 Discussion.......................................................................................................16 Chapter 3. Transdifferentiation of Human Chondrocytes into Myogenic Commitment.23 3.1 Introduction.....................................................................................................23 3.2 Methods...........................................................................................................24 3.2.1 Generation of plastic state chondrocytes.................................................24 3.2.2 Myogenic differentiation with SB-431542..............................................24 - 4 - 3.2.3 RT-PCR....................................................................................................24 3.2.4 Immunocytochemistry.............................................................................25 3.3 Results.............................................................................................................26 3.3.1 Gene expression level differences during transfection............................26 3.3.2 Morphological change..............................................................................26 3.3.3 Gene expression level differences during SB-compound treatment........26 3.4 Discussion.......................................................................................................27 Chapter 4. Conclusion............................... References....................................................................................................................... 34 요약 (국문).............. ......................................................................... 38 | - |
dc.format | application/pdf | - |
dc.format.extent | 1309327 bytes | - |
dc.format.medium | application/pdf | - |
dc.language.iso | en | - |
dc.publisher | 서울대학교 대학원 | - |
dc.subject | transdifferentiation | - |
dc.subject | poly(b-amino esters) | - |
dc.subject | minicircle DNA | - |
dc.subject | SB-431542 | - |
dc.subject.ddc | 660 | - |
dc.title | Induced myogenic commitment of human chondrocytes via non-viral delivery of minicircle DNA | - |
dc.title.alternative | 미니써클 유전체의 비바이러스성 전달을 통한 인간 연골세포의 근육세포로의 분화 유도 | - |
dc.type | Thesis | - |
dc.contributor.AlternativeAuthor | Jieun Hong | - |
dc.description.degree | Master | - |
dc.citation.pages | 54 | - |
dc.contributor.affiliation | 공과대학 화학생물공학부 | - |
dc.date.awarded | 2014-08 | - |
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