博碩士論文 103324036 詳細資訊




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姓名 林泓任(Hong-Ren Lin)  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 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
(Human adipose-derived stem cell isolation from fat tissues by membrane filtration method via nylon net filters having different pore size)
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摘要(中) 人類脂肪幹細胞是一種幹細胞,這種細胞在現在被認為是一種十分具有發展潛力的幹細胞。這是因人類脂肪幹細胞並不像人類胚胎幹細胞一樣有道德爭議。而這種幹細胞還展現了許多不同的特質,包含其良好的分化能力。
被分離出來的人類脂肪幹細胞其純度會因為分離手法而呈現許多不同層級的純度差異。而混成膜細胞遷移方法是由我們所發展出的細胞純化手法,使用這種方法可以得到高純度的幹細胞和有較高多功能性基因表現的細胞。在這方法中,我們讓剛萃取出來的脂肪幹細胞穿過多孔的薄膜,而這種材料的孔徑大小由11微米到80微米。過濾之後膜會培養在培養基中一段時間來讓其細胞數成長,並使用流式細胞儀分析各種條件下的細胞表面蛋白比例。而這些從薄膜中遷移出來的細胞,在流式細胞儀的分析之下我們發現有極高比例可以表現出間質幹細胞的
表面標誌蛋白。
另外,與傳統的分離方式相比,這種分離方式所得到的細胞也展現出較高的多功能性基因表現亮 (像是Oct4, Sox2, Klf4 和Nango)。而成骨分化檢測中,其藉由茜素紅染色所測得的鈣含量累積也較為高。
從這些方面來看,使用不同孔徑大小之耐倫薄膜所分離出來的人類脂肪幹細胞在做為再生醫學應用上展現了良好的發展潛力。
摘要(英) Human adult stem cells, such as human adipose-derived stem cells (hADSCs), are considered to be a more attractive source of stem cells than human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). This is because human adult stem cells do not generate the ethical concerns that accompany hESCs. The hADSCs exhibit heterogeneous characteristics, indicating various genotypes and differentiation abilities.
The isolated hADSCs can possess different purity levels and divergent properties depending on the purification methods used. A hybrid-membrane migration method was developed to purify hADSCs from a fat tissue solution with extremely high purity and pluripotency. A primary fat-tissue solution was permeated through the porous membranes with a pore size from 11 to 80 μm by the membrane filtration method, and the membranes were incubated in cell culture medium for 15-18 days to expand cell number and hADSCs as well as a primary fat-tissue solution were analyzed using flow-cytometry to evaluate mesenchymal stem cell surface markers. The migrated cells from the membranes were also analyzed using flow cytometry, after the membranes were cultivated in the cell culture medium after permeation of primary fat-tissue solution. The hADSCs that migrated from the membranes contained an extremely high percentage (e.g., >95%) of cells positive for mesenchymal stem cell markers.
Compared with cells isolated by conventional culture method, the cells isolated by the membrane filtration method showed higher expression of several pluripotency genes (Oct4, Sox2, Klf4 and Nanog) and more calcium accumulation evaluated by alizarin red staining (ARS) and Von Kossa staining (VKS).
hADSCs with high purity and pluripotency will be useful as a cell source for regenerative medicine. Especially, the effect of pore size of Nylon net filter membranes on purity and yield of the isolated hADSCs were investigated by the hybrid-membrane migration method and membrane filtration method in this study.
關鍵字(中) ★ 人類脂肪幹細胞
★ 純化
★ 膜
★ 耐倫
★ 孔徑大小
關鍵字(英) ★ human adipose-derived stem cell
★ purify
★ membrane
★ nylon
★ pore siez
論文目次 Chapter 1 Introduction-1
1-1 Stem Cells-1
1-1-1 Embryonic stem cells (ESCs)-2
1-1-2 Induced pluripotent stem cells (iPSCs)-2
1-1-3 Mesenchymal stem cells (MSCs)-2
1-2 Adipose-derived stem cells-3
1-3 Differentiation capacity of adipose-derived stem cells-4
1-3-1 Adipogenic differentiation-5
1-3-2 Osteogenic differentiation-5
1-3-3 Myogenic and cardiomyogenic differentiation-7
1-4 Immunophenotype-7
1-5 Isolation of adipose-derived stem cells-8
1-5-1 Cell isolation-9
1-5-2 Membrane filtration method and migration method-9
1-5-3 Magnetic-activated cell sorting (MACS)-10
1-5-4 Flow Cytometry-11
1-5-4-1 Fluorescence-activated cell sorting (FACS)-12
1-6 Polymerase chain reaction (PCR)-12

Chapter 2 Materials and Methods-14
2-1 Materials-14
2-1-1 Nylon mesh filters-14
2-1-2 Culture medium-14
2-1-3 Osteogenic induction medium-14
2-1-4 Phosphate buffer saline solution (PBS)-14
2-1-5 Digestion solution-14
2-1-6 ACK lysis solution-15
2-1-7 Flow Cytometry-15
2-1-8 RNA extraction-15
2-1-9 Reverse transcription (RT)-15
2-1-10 Real-time polymerization chain reaction (q-PCR)-15
2-1-11 Q-PCR probe-15
2-1-12 Alkaline phosphatase assay-16
2-1-13 Alizarin red staining-16
2-1-14 von Kossa staining-16
2-2 Experimental Method-16
2-2-1 Preparation of phosphate buffer saline solution (PBS)-16
2-2-2 Culture medium preparation-17
2-2-3 Isolation and culture of adipose-derived stromal cells-17
2-2-4 Culture and passaging of ADSCs-18
2-2-5 Cell density measurement-19
2-2-6 Cell purification (membrane migration method)-20
2-2-7 Differentiation of adipose tissue-derived stem cells-21
2-2-8 Surface markers analyzed by flow cytometry-21
2-2-9 Isolation of total RNA-22
2-2-10 Reverse transcription of mRNA into DNA-22
2-2-11 Quantitative real time polymerase chain reaction-23
2-2-12 Alkaline phosphatase activity-24
2-2-13 Alizarin Red staining-24
2-2-14 von Kossa staining-24
2-2-15 Quantitative analysis of osteogenesis-25

Chapter 3 Results and Discussions-26
3-1 Permeation of SVF through Nylon mesh filter having different pore size-26
3-2 hADSCs purified from SVF in conventional culture method-28
3-3 Purification of hADSCs from SVF by membrane filtration and migration methods through Nylon mesh filters having different pore sizes-32
3-3-1 Growth curve of hADSCs in SVF and migrated cells from Nylon mesh filters-32
3-3-2 Flow cytometry analysis of ADSCs migrated from Nylon mesh filters-34
3-4 Pluripotency analysis of hADSCs purified by several methods-41
3-5 The differentiation ability of hADSCs purified by hybrid membrane method into osteoblasts-46

Chapter 4 Conclusions-51

Chapter 5 Appendix Data
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指導教授 樋口亞紺(Akon Higuchi) 審核日期 2016-7-6
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