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Cell cycle regulation by ankyrin repeat-rich membrane spanning scaffold protein and size-dependent fractionation of human mesenchymal stem cells using microfluidic chip filtration : Ankyrin repeat-rich membrane spanning 단백질에 의한 세포주기 조절 기작 및 미세유체칩을 이용한 중간엽 줄기세포의 크기별 분리 연구
DC Field | Value | Language |
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dc.contributor.advisor | 장미숙 | - |
dc.contributor.author | 정희경 | - |
dc.date.accessioned | 2017-07-14T05:46:18Z | - |
dc.date.available | 2017-07-14T05:46:18Z | - |
dc.date.issued | 2017-02 | - |
dc.identifier.other | 000000142305 | - |
dc.identifier.uri | https://hdl.handle.net/10371/125143 | - |
dc.description | 학위논문 (박사)-- 서울대학교 대학원 : 치의과학과, 2017. 2. 장미숙. | - |
dc.description.abstract | As one of the adaptor proteins, ankyrin rich membrane spanning protein (ARMS/Kidins220) is highly expressed in the nervous system, such as the hippocampus, olfactory burb, and motor neurons in the spiral cord. Generally, ARMS/Kidins220 interacts with the neurotrophin, ephrin, vascular endothelial growth factor (VEGF) and glutamate receptors, which have many essential roles in the nervous system such as neuronal survival, neuronal differentiation, dendrites and synapse developments. Recent studies have reported that this protein yields sustained signal of the mitogen-activated protein kinase (MAPK) via the CrkL-C3G complex and Rap1, which are both highly expressed in melanoma. This suggests the ARMS is a potential oncogene and is involved in controlling cell cycle.
Cell proliferation is tightly controlled by cyclins and cyclin-dependent kinase (CDKs) in G1, S, M, and G2 phases. First, we observed that knockdown of ARMS/Kidins220 inhibited mouse neuroblastoma cell proliferation and resulted in a slowdown of cell cycle during G1 phase by reductions of cyclin D1 and CDK4. In addition, these decrease in cyclin D1 and CDK4 protein levels cause a subsequent reduction of pRb hyperphosphorylation which occurs at the G1/S phase transition. Moreover, we observed an upregulation of p21, a CDK4 inhibitor, with downregulation of ARMS/Kidins220. Taken together, these data suggest that p21 inhibits the kinase activity of cyclin D1-CDK4 and prevents the hyperphosphorylation of pRb, resulting in down regulation of cell cycle progression. Therefore, we report that the low ARMS/Kidins220 has a role as a signaling mediator to regulate neuroblastoma proliferation and could be worked as a potential oncogene. Human mesenchymal stem cells (hMSCs) can self-renew and differentiate into multiple cell types, which make them suitable for use in tissue engineering and cell therapy. However, their clinical application is hindered by the need for in vitro culture with growth factors in order for their expansion to reach therapeutically useful levels. Moreover, hMSCs consist of a heterogeneous population that exhibits variable morphology, a limited capacity for self-renewal, and inefficient differentiation. Thus, obtaining purified hMSCs with high potential is an important step toward increasing the efficiency of stem cell therapy. For the purpose of flow-based identification of hMSC subpopulations, optimally designed microfluidic chips were developed based on the hydrodynamic filtration (HDF) principle. In these chips, hydrodynamic effects for passive separation are combined with fluid interaction along a series of channels. Microfluidic chip design parameters resulted from complete analysis of laminar flow for flow fraction and complicated networks of main and multi-branched channels, which were validated for microfluidic cell sorting by HDF to fractionate hMSCs into three size-dependent subpopulations: small rapidly self-renewing (RS, < 25 µm) | - |
dc.description.abstract | medium spindle-shaped (SS, 25–40 µm) | - |
dc.description.abstract | and large flattened (FL, > 40 µm) cells. In our HDF chip, a virtual cut-off width (WC) boundary causes hMSCs to migrate to sidewalls, whereby they enter branch channels in response to specific ratios between main and side flows. Our results showed continuous and rapid sorting of hMSCs into three subpopulations with highly efficient recovery (>86%) and complete purity rates, and without damage to cells.
Analysis of surface marker expression revealed that RS and SS cells showed high levels of CD73, CD90, and CD105, whereas FL cells did not express CD73. Subsequently, to compare the multipotency of sorted cells, each subpopulation was induced to undergo adipogenic, osteogenic, and Schwann cell (SC) differentiation. Phenotypic and gene marker expression analyses of differentiation indicated that sorted RS and SS subpopulations showed higher potential for adipogenic, osteogenic, and Schwann cell differentiation than the FL subpopulation. Moreover, RS and SS cells displayed significantly increased expression of SC markers and growth factors such as hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF). Therefore, our results indicate that the microfluidic chip successfully sorted hMSCs based on size and target cells with high multilineage potentials were obtained from hMSCs. | - |
dc.description.tableofcontents | CHAPTER I. General Introduction 1
I. Regulation of cell cycle 1 II. Ankyrin repeat rich-membrane spanning protein 6 III. ARMS/Kidins220 and neurotrophin signaling 9 IV. ARMS/Kidins220 functions related to pathologies 11 V. Human mesenchymal stem cells 13 VI. Microfluidics 15 VII. The application of microfluidics in stem cells therapy 18 VIII. Microfluidics for size based cell separation 20 IX. Hydrodynamic filtration 24 CHAPTER II. Ankyrin repeat-rich membrane spanning (ARMS)/Kidins220 scaffold protein regulates neuroblastoma cell proliferation through cyclin D1/ CDK4 and p21 27 I. Abstract 28 II. Introduction 29 III. Materials and methods 32 1. Cell culture 32 2. Generation of ARMS/Kidins220 short hairpin RNA (shRNA) retroviral vectors and ARMS/Kidins220-knockdown Neuro2a cell lines 32 3. Cell proliferation assay 33 4. Fluorescence-activated cell sorting analysis 33 5. Immunoblotting 34 6. Statistical analysis 34 IV. Results 36 1. Regulation of neuroblastoma cell proliferation by knock- down of ARMS/Kidins220 36 2. G1 phase arrest in cell cycle by downregulation of ARMS/Kidins220 38 3. Reductions of cyclin D1 and CDK4 involved in downregulation of ARMS/Kidins220 41 4. Inhibition of hyperphosphorylation of pRb via reduction of cyclin D1 and CDK4 by downregulation of ARMS/Kidins220 43 V. Discussion 48 CHAPTER III. Sorting of human mesenchymal stem cells by applying multi-branched microfluidic chip 51 I. Abstract 52 II. Introduction 54 III. Materials and methods 67 1. Sorting chip fabrication 67 2. Sorting chip operation 69 3. Sample preparations for side flow performance and particle sorting 69 4. hMSCs culture and preparations for cell sorting 70 5. Viability and growth of hMSCs subpopulations 72 6. The surface marker expressions of hMSCs subpopulations 73 7. Adipogenic, osteogenic, and Schwann cell differentiation of sorted hMCSs 73 8. Oil Red O staining and alkaline phosphate staining 74 9. Reverse transcription-polymerase chain reaction 75 10. Image analysis and statistics 76 IV. Results 79 1. hMSCs distribution 79 2. Side flow effect on focusing and flow fractionation 79 3. Sorting of bidisperse spherical particles 84 4. Sorting of hMSCs subpopulation 86 5. Cell viability and growth of hMSCs subpopulation 91 6. Expression of surface markers in the sorted subpopulations 93 7. Differentiation of hMSCs subpopulations 95 V. Discussion 110 REFERENCES 114 ABSTRACT IN KOREAN 131 | - |
dc.format | application/pdf | - |
dc.format.extent | 6072170 bytes | - |
dc.format.medium | application/pdf | - |
dc.language.iso | en | - |
dc.publisher | 서울대학교 대학원 | - |
dc.subject | ARMS/Kidisn220 | - |
dc.subject | cell cycle | - |
dc.subject | cyclin D1 | - |
dc.subject | cyclin-dependent kinase 4 | - |
dc.subject | p21 | - |
dc.subject | Human mesenchymal stem cells | - |
dc.subject | Microfluidic sorting chip | - |
dc.subject | Hydrodynamic filtration | - |
dc.subject.ddc | 617 | - |
dc.title | Cell cycle regulation by ankyrin repeat-rich membrane spanning scaffold protein and size-dependent fractionation of human mesenchymal stem cells using microfluidic chip filtration | - |
dc.title.alternative | Ankyrin repeat-rich membrane spanning 단백질에 의한 세포주기 조절 기작 및 미세유체칩을 이용한 중간엽 줄기세포의 크기별 분리 연구 | - |
dc.type | Thesis | - |
dc.contributor.AlternativeAuthor | Heekyung Jung | - |
dc.description.degree | Doctor | - |
dc.citation.pages | 133 | - |
dc.contributor.affiliation | 치의학대학원 치의과학과 | - |
dc.date.awarded | 2017-02 | - |
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