指導教授：丁詩同臺灣大學：動物科學技術學研究所張耘萃Chang, Yun-TsuiYun-TsuiChang2014-11-282018-06-292014-11-282018-06-292014http://ntur.lib.ntu.edu.tw//handle/246246/262746根據國際糖尿病聯合會的統計，在2013年全球70億5千萬人口中有3億8千萬人患有糖尿病，其中有2千8百萬人罹患第一型糖尿病。 第一型糖尿病是一種自體免疫攻擊疾病，身體的免疫系統攻擊自身的β細胞，使得胰島素產生不足，進而無法維持血糖平衡。利用間葉幹細胞 (mesenchymal stem cells)分化複能性的幹細胞療法 (stem cell therapy)，可修復受傷的組織，為一種新興療法。然而，此種療法需要大量的幹細胞進行移植，因此，為了增進幹細胞來源，我們希望能使用脂肪幹細胞加以增殖，以生產大量的細胞功醫療之用，因為其容易取得、來源豐富且具有分化複能性的特性。 我們從豬背部脂肪的皮下脂肪組織分離出脂肪幹細胞，經過分析發現取得的脂肪幹細胞具有幹細胞表面抗原表現，例如：CD29、CD44、CD90和MHC I。這些脂肪幹細胞可被刺激分化成脂肪細胞、軟骨細胞和硬骨細胞，證明此等脂肪幹細胞具有複分化潛能。利用噻唑藍比色法 (MTT assay)及細胞計數測定細胞數目，我們發現纖維母細胞生長因子-2 (fibroblast growth factor 2, FGF2)和纖維母細胞生長因子-4 (fibroblast growth factor 4, FGF4)的添加，會增加脂肪幹細胞增殖速率。我們也發現 FGF2的添加會增進ERK磷酸化，推測FGF2可能藉由ERK這個訊息傳導路徑促進脂肪幹細胞的分裂。 再進一步將大量增生的脂肪幹細胞分化成胰島β細胞，透過螢光免疫染色法、qPCR和ELISA分析，發現此等細胞都具有分化成胰島β細胞的能力，其中又以經FGF2培養的脂肪幹細胞，其胰島相關基因insulin 表現量明顯升高。這些結果顯示，生長因子的添加可促使脂肪幹細胞的增生，同時保有其分化成胰島β細胞的能力。 綜合上述研究，脂肪幹細胞可經由添加FGF2和FGF4使得細胞大量增殖，並保有其分化成胰島β細胞的能力，此類細胞應可作為治療第一型糖尿病的細胞來源。According to the statistic from International Diabetes Federation in 2013, there is 7 billion people in the world and 28 million people suffering from type 1 diabetes (T1D) in 382 million diabetes patient. T1D is caused by an auto-immune reaction where the body’s immune system attacks the β-cells which could produce insulin to maintain blood glucose. Due to the developmental plasticity of mesenchymal stem cells (MSC), MSC-based therapeutic intervention has become a promising strategy to replace injured tissues. However, a major obstacle of applying MSC is required large amounts of MSC for transplantation. To tackle this issue, we seek for adipose-derived stem cells (ADSCs) owing to their ease of isolation, abundant sources and multipotency and would like to further enhance their proliferative ability and keep differentiation characteristics. We isolated ADSCs from porcine back fat region of subcutaneous adipose tissues (pADSCs) and showed that these ADSCs expressed the MSC surface markers including CD29, CD44, CD90 and MHC I. These pADSCs also sustained the multipotency to differentiate into adipocytes, chondrocytes and osteocytes. We cultured pADSCs in basal medium supplemented with or without growth factors such as fibroblast growth factor 2 (FGF2) or fibroblast growth factor 4 (FGF4) to determine the proliferation by MTT analysis and cell counting. Our results demonstrated that both FGF2 and FGF4 increased the proliferation of pADSCs. In our experiments, FGF2 also increased ERK phosphorylation. Therefore we speculate that FGF2 may work on ERK pathway to promote pADSC proliferation. Furthermore, FGF-supplemented pADSCs could be induced to differentiate into insulin producing cells. Determined by immunofluorescent staining and examined by confocal microscopy, quantitative PCR and ELISA, β-cells markers insulin was highly expressed in FGF-supplemented pADSCs. These results indicated that FGF2 increases pADSCs proliferation without compromising the β-cells differentiation characteristics of pADSCs. To conclude, in this study, we determined the optimal culture condition for pADSCs with either FGF2 or FGF4. These pre-conditioned pADSCs provide a potential strategy to increase pADSCs proliferation for transplantation in treating type 1 diabetes or other autoimmune diseases.口試委員會審定書 I Acknowledgements II 中文摘要 III Abstract V Content XII Index of Figures X Index of Tables XIII Chapter 1 Introduction 1 Chapter 2 Literature Review 3 2.1 The endocrine pancreas 3 2.1.1 Pancreas 3 2.1.2 Pancreatic islets 4 2.1.3 Pancreatic islet development and β-cells differentiation 5 2.1.4 Insulin secrection 8 2.2 Type I diabetes (T1D) 9 2.2.1 Introduction of type I diabetes 9 2.2.2 Mechanisms in T1D 9 2.2.3 T1D treatment and management 10 2.3 Stem cell 11 2.3.1 Introduction of stem cells 11 2.3.2 Introduction of mesenchymal stem cells 12 2.3.3 Adipose derived stem cells (ADSC) 13 2.4 Growth factors 13 2.4.1 Effect of growth factors on MSCs proliferation 13 2.4.2 Effect of growth factors on MSCs differentiation 14 Chapter 3 Material and methods 16 3.1 Isolation of porcine adipose derived stem cell 16 3.2 Flow cytometry analysis 17 3.3 Differentiation of pADSC into adipocytes, osteocytes and chondrocytes 18 3.3.1 Adipocytes 18 3.3.2 Osteocytes 18 3.3.3 Chondrocytes 19 3.4 MTT assay 20 3.5 Cell counting 20 3.6 Cells treatment with JNK and ERK inhibitors 21 3.7 Western blotting 21 3.8 Preparation of chitosan-coated culture dishes 22 3.9 Differentiation of pADSC intoβ-cells 22 3.10 Immunofluorescence analysis 23 3.11 Analysis of mRNA gene expression by real-time qPCR 23 3.12 ELISA for insulin secretion 25 3.13 Statistical analysis 26 Chapter 4 Results 27 1. Characterization of adipose-derived stem cells 27 1.1 Cell surface markers for adipose derived stem cells 27 1.2 pADSCs sustained the multipotency of differentiation 29 2. Enhancing pADSC proliferation 30 2.1 Optimal growth factors for pADSC proliferation 30 2.2 FGF2 promote cell proliferation in different passages 32 2.3 FGF2 promotes ERK/JNK pathway phosphorylation 33 2.4 Stemness gene expression were decreased with increase of passages 35 3. Maintain pADSC differentiation intoβ-cells 35 3.1 Differentiation morphology in different passages 35 3.2β-cell differentiation-associated genes were decreased with increase of passages 36 3.3 Representative immunofluorescence analysis of β-cell differentiation-associated markers in different passages 38 3.4 The effect of growth factors on the expression of β-cell differentiation-associated gene 40 3.5 Glucose stimulate insulin secretion in passage 1, 3, and 5 43 Chapter 5 Discussions 46 Conclusion 49 References 503423793 bytesapplication/pdf論文公開時間：2015/08/25論文使用權限：同意有償授權(權利金給回饋學校)第一型糖尿病脂肪幹細胞纖維母細胞生長因子生長速率胰島β細胞[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/262746/1/ntu-103-R01626014-1.pdf