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研究生: 盧珍妏
Lu, Chen-Wen
論文名稱: 以氧化鐵奈米粒子標記人類間質幹細胞誘導成為類神經細胞的追蹤表現及應用
Characterization of an iron oxide nanoparticle labelling and MRI-based protocol for inducing human mesenchymal stem cells into neural-like cells
指導教授: 吳忠信
Wu, Chung-Hsin
學位類別: 博士
Doctor
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2017
畢業學年度: 105
語文別: 英文
論文頁數: 69
中文關鍵詞: 氧化鐵奈米粒子核磁共振成像間質幹細胞類神經細胞動作電位
英文關鍵詞: iron oxide nanoparticle (ION), magnetic resonance imaging (MRI), mesenchymal stem cells (MSCs), neural-like cells (NCs), action potential
DOI URL: https://doi.org/10.6345/NTNU202202909
論文種類: 學術論文
相關次數: 點閱:125下載:7
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  • 本研究的目的是以開發氧化鐵奈米粒子(ION)標記人類間質幹細胞 (MSCs)誘導體外分化成為類神經細胞 (NCs) 的應用以及在核磁共振成像(MRI)之追蹤表現。Ferucarbotran,一種臨床所使用的氧化鐵奈米粒子,此種陰性顯影劑可以在核磁共振成像下清楚的看見,因此被用來標記細胞內的追蹤觀察。本研究透過光學顯微鏡下發現體外培養的類神經細胞具有神經細胞的型態以及量測動作電位的功能表現。在光學顯微鏡下觀察到細胞呈現軸突樣的結構型態。這些類神經細胞比未分化的間質幹細胞表現較多頻率的動作電位。以氧化鐵奈米粒子標記對間質幹細胞的形態、功能和分化能力沒有影響。我們的結論發現,以體外誘導人類間質幹細胞 (MSCs)分化成的類神經細胞 (NCs) 表現較多頻率的動作電位,或許這些體外誘導生成的類神經細胞可以用於替代損傷的神經元。

    The aim of the current study was to develop an iron oxide nanoparticle (ION) labelling and magnetic resonance imaging (MRI)-based protocol to allow visualization of the differentiation process of mesenchymal stem cells (MSCs) into neural-like cells (NCs) in vitro. Ferucarbotran, a clinically available ION, which can be visualized under MRI, is used for tracking cells implanted in vivo. The NCs were verified morphologically and histologically by light microscopy, and their functions were verified by measuring their action potentials. Conformational conversion of axon-like structures was observed under light microscopy. These NCs exhibited frequent, active action potentials compared with cells that did not undergo neural differentiation. The labelling of ION had no influence on the morphological and functional differentiation capacity of the MSCs. We conclude that the MSCs that were differentiated into NCs exhibited in vitro activity potential firing and may be used to replace damaged neurons.

    TABLE OF CONTENTS I LIST OF FIGURES IV 中文摘要 V ABSTRACT VI CHAPTER 1 INTRODUCTION 1 1.1 HMSCs 2 1.2 Iron oxide nanoparticles (IONs) 3 1.3 Research aims 4 CHAPTER 2 MATERIALS AND METHODS 5 2.1 Cell culture (1) Human mesenchymal stem cell culture (2) Neurogenic differentiation of human mesenchymal stem cells (3) ION labelling 6 2.2 Morphological analysis (1) Transmission electron microscopy (2) Co-staining with Prussian Blue and phosphotungstic acid haematoxylin (PTAH) 7 2.3 Reverse transcription polymerase chain reaction (RT-PCR) 8 2.4 Western blotting 9 2.5 Immunofluorescence staining 10 2.6 Flow cytometry analysis of neural markers 11 2.7 Electrophysiological recording 11 2.8 Flow cytometry detection of ION particle uptake 12 2.9 Magnetic resonance imaging (MRI) 12 2.10 Intracellular iron content determination 13 2.11 Viability assay 13 2.12 Reactive oxygen species measurements 14 2.13 Mitochondria membrane potential measurements 14 2.14 Statistical analysis 15 CHAPTER 3 RESULTS 16 3.1 Differentiated human MSCs exhibited neural-like morphology and neuron markers: Directly labelling hMSCs and NCs with ION 17 3.2 Electrophysiological function 18 3.3 In vitro determination of ION uptake by MRI, inductively coupled plasma mass spectrometry (ICP-MS) and flow cytometry 19 3.4 Cell behavior 20 CHAPTER 4 DISCUSSION 21 4.1 Cell behavior 22 4.2 Different sizes of supraparamagnetic ION 23 4.3 NPs coating 26 4.4 Iron Content 29 4.5 Neural induction medium (NIM) 29 4.6 NCs 30 4.7 Neural Protein expression 31 4.8 Spontaneous firing frequency 32 CHAPTER 5 CONCLUSION 34 CHAPTER 6 REFERENCES 36 CHAPTER 7 FIGURES 57 CHAPTER 8 Tables 69 APPENDIX 1: CV of Chen-Wen, Lu i APPENDIX 2: Copy of Published Sci Papers ii FIGURES 57 Figure 1. Comparison of hMSC differentiation capacity into NCs with or without (w/o) ION 58 Figure 2. TEM images of ION (Resovist, ferucarbotran) 60 Figure 3. Characterization of neural differentiation markers in hMSCs treated with or without neural induction medium after ION labelling 61 Figure 4. Action potentials of hMSCs, NCs with or without ION labelling 63 Figure 5. Quantification of iron content after labelling with or without ION before and after induction of neural-like cell differentiation 65 Figure 6. Measuring cell behaviour using three different assays 67 Table 1. Primer sequences used for RT-PCR analysis 69

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