博碩士論文 993204047 詳細資訊




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姓名 吳承翰(Chen-Han Wu)  查詢紙本館藏   畢業系所 化學工程與材料工程學系
論文名稱 人類脂肪幹細胞的膜純化法與分化能力研究
(Purification and Differentiation of Human Adipose-derived Stem cells by Membrane Filtration Method)
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★ 人類體細胞從組成誘導型多能性幹細胞培養在無飼養層上★ 使用不同孔洞大小之耐倫薄膜從脂肪組織中分離及純化人類脂肪幹細胞之研究
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摘要(中) 脂肪幹細胞對於再生醫學生上是個很棒的幹細胞來源,由於它具有大量且低手術傷害性取得等優點。此外,脂肪幹細胞具有分化成中胚層細胞的能力,像是骨細胞、軟骨細胞、肌肉細胞等。甚至能分化成外胚層的細胞,像是神經細胞。因此脂肪幹細胞在組織再生醫學與組織工程學上被視為極具潛力的幹細胞來源。
目前組織純化細胞的方法最傳統的方式為細胞培養法,然後這種方法需要長時間的培養與繼代。螢光活性細胞分選 (FACS)和磁力活性細胞分選(MACS)也常選用作為組織細胞的純化萃取,但這種利用抗體專一性分離的方法,抗體可能有病毒汙染和高成本的疑慮。膜分離法具有簡單快速等優點,且分離過程在完全無汙染的環境中。此項研究試圖從人類脂肪組織中萃取出含有SVF的初代細胞溶液中,快速萃取出可表現脂肪幹細胞表面標誌且具有分化能力細胞。
我們使用傾注式過濾法,由於此法有一開口可宣洩壓力,不會推擠細胞通過過濾膜。另外準備5μm、12μm和白血球細胞移除膜(leukocyte removal filter)等具有不同孔洞大小的複雜結構聚氨酯膜。在初代代細胞通過過濾膜後所得之細胞溶液稱之為過濾液,然後再由相反方向注入培養液所得到未能通過膜的細胞之細胞溶液稱之為回收液。藉由流式細胞儀分析細胞表面標誌和測試細胞的分化能力,其結果顯示出12μm 孔洞大小的聚氨酯膜具有較佳的分離效果。另外我們也測試經表面改質過後的聚氨酯膜,但結果顯示經改質過後的膜具有較強的幹細胞吸附性,降低了分離的效果。
摘要(英) Adipose-derived stem cells (ADSCs) are a promising cell source in regenerative medicine, of particular utility for cell therapies and tissue engineering, because adipose tissue can easily be harvested in large quantities compared to bone marrow, and ADSCs have high proliferation rates in culture. ADSCs are isolated from adipose tissue by liposuction and centrifugation followed by cultivation on cell culture dishes for at least one passage. The cultivation of cells derived from adipose tissue is necessary to purify ADSCs (i.e., “the culture method” for the purification of ADSCs) because the adipose tissue contains not only ADSCs but also adipose and other types of cells. The culture process for the purification of ADSCs requires several days, at minimum. If ADSCs can be purified from adipose tissue in a short period of time (i.e., less than 2 hrs) by using a cell purification device such as the membrane filtration method, cell therapy and tissue engineering applications using autologous ADSCs might become more efficient.
Therefore, we investigated the purification of human ADSCs from a digested solution of adipose tissue by the membrane filtration method in this study, and we compared the purity of ADSCs and the differentiation ability of ADSCs into osteoblasts after purification by the membrane filtration method and the conventional cell culture method.
We investigated two filtration methods to purify hADSC, i.e., batch-type and perfusion-type filtration methods. Main differences between these two filtration methods are cell flow direction to the membranes. Polyurethane foaming membranes having 5-12 μm of pore size were used as the membranes for the separation of hADSCs from human adipose tissue. The surface marker of ADSCs (e.g., CD73 and CD90) in the cells in the permeate and recovery solutions were analyzed by flow cytometry whether the mesenchymal stem cells were enriched after permeation through the membranes. The differentiation of cells into osteoblasts, which were separated by the membrane filtration method was evaluated to confirm the enriched hADSC in the permeate solution through the membranes by culture of the cells in induced medium of osteogenic differentiation.
We, further, investigated the isolation of ADSCs by the membrane filtration method through surface-modified PU membranes having with various nanosegments (e.g., -NH2, -SO3H, -OH, and -COOH) and ECMs, and compared the isolation efficiency of ADSCs purified through nonmodified PU membranes and surface-modified PU membranes. We found that the cells separated through PU membranes by the perfusion method showed high popuration of ADSCs from surface marker analysis and the highest osteogenic differentiation ability.
關鍵字(中) ★ 間葉幹細胞
★ 成骨分化
★ 人類脂肪幹細胞
★ 膜純化法
關鍵字(英) ★ Human Adipose-derived Stem Cells
★ Membrane Filtration method
★ Mesenchymal stem cells
★ Osteogenic differentiation
論文目次 摘要 ....................................................................................................................................................... I
Index of Contents .............................................................................................................................. IV
Index of Figures ...............................................................................................................................VII
Index of Tables ............................................................................................................................... XIV
Chapter 1. Introduction ...............................................................................................................1
1-1 Stem Cells ............................................................................................................................1
1-2 Adipose-derived stem cells (ADSCs).................................................................................2
1-3 Cell molecular marker of adipose-derived stem cell .......................................................4
1-3.1 Immunophentype ........................................................................................................4
1-3.2 Gene expression of ADSCs .........................................................................................7
1-4 Isolation of adipose-derived stem cells .............................................................................8
1-4.1 Cell isolation ...............................................................................................................8
1-4.2 Membrane Purification ..............................................................................................9
1-5 Differentiation capacity of adipose-derived stem cells ..................................................10
1-5.1 Lineage-specific differentiation potential ................................................................10
1-5.2 Adipogenic differentiation ........................................................................................ 11
1-5.3 Chondrogenic/Osteogenic differentiation ................................................................12
1-5.4 Myogenic/cardiomyogenic differentiation ...............................................................14
1-5.5 Other effect factor of MSC differentiation ..............................................................15
1-6 Extracellular matrix .........................................................................................................16
1-6.1 Collagen (Col) ...........................................................................................................16
1-6.2 Fibronectin (FN) ......................................................................................................19
1-7 Flow cytometry .................................................................................................................20
1-8 Polymerase chain reaction (PCR) ...................................................................................23
1-8.1 Introduction of PCR .................................................................................................23
1-8.2 The procedure of PCR ..............................................................................................24
Chapter 2. Materials and Methods ...........................................................................................27
2-1 Materials .............................................................................................................................27
2-1.1 Chemicals ..................................................................................................................27
2-1.2 Consumable apparatus ..............................................................................................28
2-1.3 Instruments ................................................................................................................29
2-2 Experimental Methods .......................................................................................................30
2-2.1 PBS (phosphate buffer saline solution) preparation .................................................30
2-2.2 Culture medium preparation .....................................................................................30
2-2.3 Cell culture and passages ..........................................................................................30
2-2.4 Isolation and culture of adipose tissue-derived stromal cell .....................................32
2-2.5 Preparation of surface-mdified PU foaming membranes ..........................................33
2-2.5.1 Coating method .....................................................................................................34
2-2.5.2 Chemical grafting method .....................................................................................34
2-2.6 Cell purification (membrane method) .......................................................................37
2-2.7 Differentiation of adipose tissue-derived Stem cell ...................................................38
2-2.8 Immunology staining .................................................................................................38
2-2.9 Alizarin red S staining ...............................................................................................38
2-2.10 von Kossa staining ................................................................................................39
2-2.11 Quantitative analysis of osteogenesis ...................................................................39
2-2.12 Alkaline phosphatase activity ................................................................................40
2-2.13 Isolation of total RNA ............................................................................................40
2-2.14 Reverse Transcription of mRNA into cDNA ..........................................................41
2-2.15 PCR (Polymerase Chain Reaction) .......................................................................43
2-2.16 Scanning electron microscopy (SEM) analysis .....................................................44
Chapter 3. Results and Discussion ............................................................................................45
3-1 Surface characterization of PU membrane .........................................................................45
3-2 Characterization of human adipose derived stem cells (hADSC) ......................................48
3-2.1 Cell culture method....................................................................................................48
3-3 Purification of adipose tissue-derived stem cells by membrane filtration method .............51
3-3.1 Effect of different pore size of PU membrane ............................................................52
3-3.1.1 Flow cytometry analysis of human adipose tissue solution ..................................52
3-3.1.2 The differentiation ability of ADSC purified by different pore size of PU membranes
and different commercial membranes .........................................................................................57
3-3.2 Effect of functional groups of 12μm PU membranes on purification of hADSCs .....60
3-3.2.1 Flow cytometry analysis of human adipose tissue solution ..................................60
3-3.2.2 The differentiation ability of cells in recovery solution through 12μm PU
membrane having different functional groups ............................................................................65
3-3.3 Effect of 12μm PU membrane having ECMs .............................................................72
3-3.3.1 Flow cytometry ......................................................................................................72
3-3.3.2 The differentiation ability of cells in recovery solution through 12μm PU
membrane coated with ECMs .....................................................................................................75
Chapter 4. Conclusion ................................................................................................................81
Supplementary data ..........................................................................................................................83
Reference ............................................................................................................................................87
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指導教授 樋口亞紺(Akon Higuchi) 審核日期 2012-7-9
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