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研究生:李俊賢
研究生(外文):LI, JYUN-XIAN
論文名稱:利用聚乙烯醇取代血清白蛋白於建立人類造血幹細胞無血清增殖培養基之研究
論文名稱(外文):Using Polyvinyl Alcohol to Replace Serum Albumin to Develop a Serum-Free Expansion Medium for Human Hematopoietic Stem Cells
指導教授:姚少凌
指導教授(外文):YAO, CHAO-LING
口試委員:朱一民簡志青
口試委員(外文):CHU, I-MINGCHIEN, CHIH-CHING
口試日期:2021-06-28
學位類別:碩士
校院名稱:元智大學
系所名稱:化學工程與材料科學學系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2021
畢業學年度:109
語文別:中文
論文頁數:74
中文關鍵詞:聚乙烯醇
外文關鍵詞:Polyvinyl Alcohol
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  • 被引用被引用:0
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造血幹細胞(Hematopoietic stem cell, HSC)是具有分化多能性與自我更新能力的一種成體幹細胞,在臨床上可用來進行骨髓造血功能重建、治療免疫缺陷、白血病…等多種疾病,但由於造血幹細胞數量極為稀少,限制了其在研究上的發展與醫療上的用途,因此如何在體外建立造血幹細胞有效培養擴增一直是困擾基礎研究和臨床應用的難題。本實驗室過去成功地開發出一種體外增殖造血幹細胞的無血清培養基,內涵4種血清取代物(1)胎牛血清白蛋白(Bovine serum albumin, BSA)、(2)胰島素、(3)轉鐵蛋白質以及(4)2-巰基乙醇。其中BSA為一種分子量不明確之動物性蛋白,不利於後續的臨床應用,而日前有文獻指出,聚乙烯醇(Polyvinyl alcohol, PVA,一種高分子)具有取代血清白蛋白的可行性。因此本實驗主要目的是利用高分子聚合物建立一種人類造血幹細胞化學成分確定之無血清增殖培養基,希望開發出一種取得更容易、成本更便宜的無血清培養基。
在本研究中,在添加血清白蛋白以及不同分子量與濃度的聚乙烯醇之無血清培養基中,將造血幹細胞進行七天的增殖培養,增殖後的細胞再進行一系列功能性分析與比較,如幹細胞增殖倍數、表面抗原分析、群落形成單位分析以及即時聚合酶鏈式反應等實驗,確認具有正確的造血幹細胞的特性。而目前的實驗結果可以很明確的知道,無血清培養基在以PVA取代的情況下增殖倍數能夠達到BSA的一半以上,證明了以高分子聚合物去替換原始的血清白蛋白是可行的,並且在PVA添加濃度為1.0 mg/ml以及分子量為Mw 13,000~23,000 Da時,有最佳的增殖倍數,另外增殖七天之後的造血幹細胞表現出了與原始造血幹細胞相似的CD34高表現量和CD38低表現量,初步確立依然擁有造血幹細胞的特性。而在群落形成單位分析的結果則顯示,在以不同PVA分子量取代原始血清白蛋白的情況下,群落形成的數量有略為高出於BSA以及HSA,且在各個類型的群落百分比能看到在CFU-GEMM也是高於BSA以及HSA的,表示與前面的實驗結果一致,HSC依然保有原有的特徵。而在添加不同濃度的5β-THB時,在群落形成的數量以及群落百分比中CFU-GEMM的比例,都是明顯低於BSA以及HSA,證實了5β-THB確實能夠讓幹細胞促進分化而不是增殖。
綜合上述,本研究旨在建立一種人類造血幹細胞化學成分確定之無血清增殖培養基,能夠以更少的資源和成本,提供醫療以及應用方面更普遍的造血幹細胞增殖方法。

Hematopoietic stem cells (HSCs) are a kind of adult stem cell with differentiation, pluripotency and self-renewal ability. It can be used clinically to reconstruct hematopoietic function of bone marrow after treat immunodeficiency, leukemia and lymphoma. However, the scarcity of HSCs limits its research and medical application. Hence, it is an important issue to establish an effective expansion culture system of HSCs in vitro for basic research and clinical applications. In the past, our laboratory successfully developed a serum-free medium for in vitro proliferation of HSCs, which contains 4 kinds of serum substitutes (bovine serum albumin, insulin, transferrin and 2-mercaptoethanol). Bovine serum albumin (BSA) is a protein from animal source with high cost and undefined molecular weight, which is not suitable for clinical applications. Recently, some literature demonstrated that a polymer, Polyvinyl alcohol (PVA) showed the role of serum albumin in the cell culture. Therefore, the aim of this study is to use PVA-like polymers to establish a low cost, chemical defined and serum-free proliferation medium for HSCs.In this study, we designed several serum-free media supplemented with different serum albumin, different molecular weight and concentrations of PVA. After 7-day culture in these media, the functional assays of expanded HSCs will be checked to confirm the stemness of expanded cells, including the cell expansion fold, surface antigen expression, colony forming unit ability and real-time polymerase chain reaction assay. The results showed that we could clearly know that the cell expansion fold of the serum-free media supplemented with PVA could reach more than half of BSA. It proved that it is feasible to replace the serum albumin with polymers. And when the concentration of PVA is 1.0 mg/ml, molecular weight of PVA is 13,000~23,000 Da, we could have the best expansion fold. In addition, HSCs cultured after 7-day showed high CD34 expression and low CD38 expression, which is similar to primitive HSC, and it was initially known that they still have the characteristics of HSC. The result of colony forming unit assay showed that when we used different molecular weight of PVA to replace serum albumin, its colony numbers was slightly higher than BSA and HSA, and the percentage of CFU-GEMM was also higher than those. It means that it’s consistent with the previous experimental results, and HSC still retains its characteristics. Finally, when we added different concentrations of 5β-THB, the colony numbers of and the percentage of CFU-GEMM were significantly lower than BSA and HSA, confirming that 5β-THB could promote stem cell to differentiate, not proliferate.
In summary, the aim of this study is to establish a chemical defined and serum-free medium for human HSC expansion in vitro, which can provide a low cost and promising method to expand the number of HSCs for medical treatment and application.
摘要 I
Abstract IV
目錄 VI
圖目錄 X
表目錄 XII
縮寫對照 XIII
第一章 緒論 1
1.1前言 1
1.2研究動機與目的 2
1.3研究架構 3
第二章 文獻回顧 4
2.1幹細胞(Stem cell)簡介 4
2.1.1何謂幹細胞 4
2.1.2幹細胞的分類 5
2.2造血幹細胞(Hematopoietic stem cell, HSC)簡介 7
2.2.1何謂造血幹細胞 7
2.2.2造血系統 8
2.2.3造血幹細胞的鑑定 10
2.3造血幹細胞的體外增殖 12
2.3.1無血清培養基 12
2.3.2基礎培養基 (basal medium) 14
2.3.3血清取代物 (Serum substitutions) 15
2.3.4細胞激素 (Cytokines) 16
2.4造血幹細胞的檢測方法 17
2.4.1群落形成單位分析 (colony forming unit analysis) 17
2.4.2表面抗原分析 (surface antigens analysis) 18
2.4.3 原始細胞的長期培養 (long-term culture-initiating cell, LTC-IC) 18
2.5 造血幹細胞的臨床應用 18
2.6 造血幹細胞臨床應用上的瓶頸 19
2.7聚乙烯醇(Polyvinyl alcohol, PVA)簡介 20
2.7.1聚乙烯醇的性質 20
2.7.2聚乙烯醇的用途 21
2.7.3聚乙烯醇在生物化學上的應用 22
2.7.4聚乙烯醇的選擇 23
第三章 實驗儀器與藥品 24
3.1實驗儀器與材料 24
3.2實驗藥品 25
3.2.1培養基/血清取代物/細胞激素 25
3.2.2流式細胞儀抗體/試劑 26
3.2.3其他藥品 27
第四章 實驗步驟與方法 28
4.1細胞培養基礎技術與注意事項 28
4.2臍帶血(Cord blood)中分離純化出CD133+造血幹細胞(HSC) 29
4.2.1單核球細胞(Mononuclear cell, MNC) 29
4.2.2臍帶血(Cord blood)中分離純化出CD133+造血幹細胞(HSC) 31
4.3細胞體外培養 32
4.4不同的培養基條件 34
4.4.1聚乙烯純(PVA)的分子量的比較 34
4.4.2血清白蛋白的替換 35
4.4.3改變聚乙烯醇(PVA)的添加濃度 36
4.5聚乙烯醇(PVA)水溶液的製備 37
4.6細胞計數與活細胞數計算 38
4.7細胞表面抗原分析(Cell surface antigen analysis) 39
4.7.1螢光抗體接合步驟 39
4.7.2流式細胞儀分析步驟 40
4.8群落形成單位分析(colony forming units analysis) 40
4.8.1 HSC種植步驟 40
4.8.2不同條件的比較 41
第五章 實驗結果與討論 42
5.1細胞型態 42
5.2確立實驗對照組 43
5.3 PVA不同分子量的比較 43
5.3血清白蛋白替換的比較 46
5.4 PVA不同濃度添加的比較 49
5.5表面抗原分析 52
5.6群落形成單位分析 60
第六章 結論與未來工作 64
6.1結論 64
6.2未來工作 65
參考文獻 67

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