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研究生:姚少凌
研究生(外文):Chao-Ling Yao
論文名稱:造血幹細胞體外增殖培養技術與應用
論文名稱(外文):Ex vivo expansion of hematopoietic stem cells
指導教授:朱一民朱一民引用關係
指導教授(外文):I-Ming Chu
學位類別:博士
校院名稱:國立清華大學
系所名稱:化學工程學系
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:2004
畢業學年度:92
語文別:中文
論文頁數:113
中文關鍵詞:造血幹細胞臍帶血無血清培養基體外增殖
外文關鍵詞:hematopoietic stem cellumbilical cord bloodserum-free mediumex vivo expansion
相關次數:
  • 被引用被引用:10
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本實驗的主要目的是在開發適合臍帶血中造血幹細胞的體外增殖無血清培養基,希望克服臍帶血在臨床移植應用上數量過少的缺點。實驗首先將新生兒的臍帶血進行分離與純化的步驟,可以得到單核細胞(mononuclear cell,MNC)與高均一性的CD133+ cell。接著利用2階因子的實驗設計方法來篩選造血幹細胞體外增殖所需要的細胞激素與血清取代物組成,並搭配著陡升路徑的實驗來找尋細胞激素與血清取代物的最適化濃度,之後再改變各種不同的基礎培養基,最後開發出兩種適合造血幹細胞增殖的無血清培養基,分別為SF-HSC與SF-MNC。SF-HSC的組成為Iscove’s modified Dulbecco’s medium (IMDM)內添加細胞激素配方CC-S6 (8.46 ng/ml TPO、4.09 ng/ml IL-3、15 ng/ml SCF、6.73 ng/ml FL、0.78 ng/ml IL-6 、3.17 ng/ml G-CSF 與1.30 ng/ml GM-CSF)與血清取代物配方BIT2 (1.5 g/l BSA、4.39 μg/ml insulin、60μg/ml transferrin與25.94 μM 2-ME)。CD133+ cell在SF-HSC中批次培養7天可達最佳的增殖效果,white blood cell (WBC)、CD34+ cell、CD34+CD38- cell、colony-forming unit cell (CFU) cell與long-term culture-initiating cell (LTC-IC)的增殖倍數分別為64.2倍、27.4倍、72.4倍、22.2倍與72.4倍。SF-MNC的組成為IMDM內添加細胞激素配方CC-S9 (5.53 ng/ml TPO、2.03 ng/ml IL-3、16 ng/ml SCF、4.43 ng/ml FL、2.36 ng/ml IL-6 、1.91 ng/ml G-CSF 、1.56 ng/ml GM-CSF、2.64 ng/ml SCGF與0.69 ng/ml IL-11)與血清取代物配方BIT (4 g/l BSA、0.71 μg/ml insulin與27.81μg/ml transferrin)。MNC在SF-MNC中批次培養6天可達最佳的增殖效果,WBC、CD34+ cell、CD34+CD38- cell、CFU cell與LTC-IC的增殖倍數分別為1.4倍、30.4倍、63.9倍、10.7倍與2.8倍。增殖後的細胞都具有端粒酶活性。
若實驗將培養基的添加策略改為一星期更換兩次,則 WBC與CD34+ cell可以持續的培養達10個禮拜以上,而且細胞的數目都能夠持續的增加。上述的這些培養效果都優於其他的商業培養基,同時實驗所開發的培養基內所含的細胞激素的濃度是其他文獻所提到培養基與商業培養基配方的十分之一,因此證明所開發的SF-HSC與SF-MNC是具有經濟效益與應用潛力的造血幹細胞體外增殖培養基。
The development of ex vivo culture systems that facilitate the expansion of hematopoietic stem and progenitor cells is crucial to stem cell research and clinical application. In this study, two serum-free, stroma-free and cytokine-containing culture media (SF-HSC and SF-MNC) for white blood cells (WBC), CD34+ cells, CD34+CD38- cells, colony-forming unit cells (CFU cells), and long-term culture-initiating cells (LTC-IC) expansion were systematically developed and optimized using the two-level factorial design and steepest ascent methods. The experimental results showed that the optimal compositions of the serum substitutes and the cytokine cocktail in SF-HSC were BIT2 (1.5 g/L BSA, 4.39 μg/mL insulin, 60 μg/mL transferrin, and 25.94 μM 2-ME), and CC-S6 (8.46 ng/mL TPO, 4.09 ng/mL IL-3, 15 ng/mL SCF, 6.73 ng/mL FL, 0.78 ng/mL IL-6, 3.17 ng/mL G-CSF, and 1.30 ng/mL GM-CSF) in the Iscove’s modified Dulbecco’s medium (IMDM), respectively. After one-week culture, the increases in the total number of WBC, CD34+ cells, CD34+CD38- cells, CFU cells, and LTC-IC were 64.2-, 27.4-, 72.4-, 22.2-, and 8.1-fold,respectively. The experimental results also showed that the optimal compositions of the serum substitutes and the cytokine cocktail in SF-MNC were BIT (4 g/l BSA, 0.71 μg/ml insulin, and 27.81μg/ml transferring), and CC-S9 (5.53 ng/ml TPO, 2.03 ng/ml IL-3, 16 ng/ml SCF, 4.43 ng/ml FL, 2.36 ng/ml IL-6, 1.91 ng/ml G-CSF, 1.56 ng/ml GM-CSF, 2.64 ng/ml SCGF, and 0.69 ng/ml IL-11) in the IMDM, respectively. After 6-day culture, the increases in the total number of WBC, CD34+ cells, CD34+CD38- cells, CFU cells, and LTC-IC were 1.4-, 30.4-, 63.9-, 10.7-, and 2.8-fold,respectively.
If SF-HSC or SF-MNC were changed twice per week, WBC and CD34+ cell could grow up continuously for at least 10 weeks. Its expansion ability of CD34+ cells and CFC was superior or comparable to that of X-vivo 20TM, StemlineTM, and StemspanTM commercial media. Furthermore, SF-HSC and SF-MNC has the lowest concentration of cytokine cocktail. These systematic methodologies are helpful in improving the ex vivo expansion system for hematopoietic stem cell and progenitor cells.
目 錄

第一章 研究動機與目的 1
1.1 楔子
1.2 研究動機與目的
1.3 研究架構
1
2
3
第二章 文獻回顧 5
2.1幹細胞(stem cell)簡介 5
2.1.1 幹細胞研究發展的起源 5
2.1.2 何謂幹細胞 6
2.1.3 幹細胞的命運(fate) 7
2.1.4 幹細胞的分類 7
2.1.5 幹細胞的應用與前景 9
2.1.6 幹細胞的來源 11
2.2 造血幹細胞(hematopoietic stem cell, HSC)簡介 17
2.2.1 何謂造血幹細胞與造血系統 17
2.2.2 造血幹細胞的起源 19
2.2.3 造血幹細胞的鑑定 20
2.2.4 造血幹細胞的臨床應用與未來發展 22
2.2.5 造血幹細胞在臨床應用上的瓶頸 24
2.3 造血幹細胞的體外增殖( ex vivo expansion) 25
2.3.1 造血幹細胞體外增殖的瓶頸 25
2.3.2 造血幹細胞體外增殖培養的方法 26
2.3.3 細胞激素的種類 27
2.3.4血清取代物的種類 29
2.4 造血幹細胞的檢測方法 32
2.5 實驗設計與統計分析的方法 34
第三章 實驗步驟與方法 36
3.1 培養動物細胞之基本技術 36
3.2 人類臍帶血的收集 37
3.3 紅血球的去除與單核細胞(mononuclear cell, MNC)的獲得 37
3.4 CD34+ cell的富集 38
3.5 細胞培養(cell culture) 40
3.6 細胞濃度與存活率計算 40
3.7 細胞群落形成單位分析(CFU assay) 41
3.8原始細胞的長期培養分析(LTC-IC assay) 42
3.8.1 Feeder layer的製備 42
3.8.2 LTC-IC assay 43
3.9 細胞表面抗原分析(cell surface antigen analysis) 43
3.9.1 螢光抗體接合步驟 43
3.9.2 FACS Calibur analyzer分析步驟 44
3.10 端粒酶活性相關分析 45
3.10.1 端粒酶RT-PCR分析(telomerase RT-PCR analysis) 45
3.10.1.1 RNA萃取 45
3.10.1.2 RT-PCR amplification 46
3.10.1.3 DNA電泳分析 47
3.10.2 端粒酶活性分析(telomerase activity analysis) 47
3.10.2.1 蛋白質萃取 47
3.10.2.2 PCR (polymerase chain reaction) amplification 48
3.10.2.3 Page analysis 48
3.11 細胞分泌激素分析(secretion cytokine analysis) 49
3.12 實驗設計與統計分析 50


第四章 實驗材料與設備 51
4.1 實驗材料與儀器 51
4.2 實驗藥品 52

第五章 結果與討論 54
5.1 臍帶血特性分析 56
5.2 開發CD133+ cell的無血清培養基 56
5.2.1 在含血清的培養基中篩選細胞激素並最適化其濃度 56
5.2.2 在IMDM+TISF的培養基中篩選血清取代物
並最適化其濃度 59
5.2.3 在IMDM+BIT2的培養基中再次篩選細胞激素
並最適化其濃度 62
5.2.4 BIT2與CC-S6在各種基礎與商業培養基中的比較 64
5.2.5 造血幹細胞在SF-HSC中的生長曲線 66
5.2.6 SF-HSC培養基與IMDM+10% FBS+TISF培養基
的比較 67
5.2.7 造血幹細胞在SF-HSC中長期的培養 67
5.3開發MNC的無血清培養基 68
5.3.1 在IMDM+CC-S9培養基中篩選血清取代物並最適化其濃度 68
5.3.2 在IMDM+BIT的培養基中篩選細胞激素並最適化其濃度 70
5.3.3 BIT與CC-S9在各種基礎與商業培養基中的比較 72
5.3.4 MNC在SF-MNC中的生長曲線 74
5.3.5 MNC在SF-MNC中長期的培養(long-term culture) 75
5.4 端粒酶活性分析 76
5.5 在SF-HSC與SF-MNC中培養後的細胞所分泌的細胞激素 77
5.6 Scale-up 79

第六章 結論與未來展望 101
6.1 結論 101
6.2 未來展望 102

第七章 參考文獻 104

附錄一 作者介紹 112
附錄二 著作 113





















圖 目 錄

圖1.1 研究架構與流程 4
圖2.1 胚胎發育的過程 9
圖2.2 造血幹細胞分化的途徑 18
圖3.1 buffy coat cells經過Ficoll-Paque分離步驟後的結果 38
圖3.2 Magnetic column與Miltenyi VarioMACS device 39
圖3.3 群落形成培養結果 41
圖3.4 FACS Calibur analyzer 45
圖5.1 臍帶血的特性與其中所含的細胞 81
圖5.2 臍帶血中(A) MNC與(B) CD133+ cell的表面抗原分析 81
圖5.3 在IMDM+10% FBS中4種細胞激素的陡升路徑及結果 82
圖5.4 在IMDM+TISF中4種血清取代物的陡升路徑及結果 82
圖5.5 在IMDM+BIT2中7種細胞激素的陡升路徑及結果 83
圖5.6在SF-HSC中WBC與CD34+ cell的生長曲線 83
圖5.7在SF-HSC與含血清培養基中WBC與CD34+ cell
的生長曲線 84
圖5.8在SF-HSC中WBC與CD34+ cell長期培養的結果 84
圖5.9在IMDM+CC-S9中3種血清取代物的陡升路徑及結果 85
圖5.10 在IMDM+BIT中9種細胞激素的陡升路徑及結果 85
圖5.11 在SF-MNC中WBC與CD34+ cell的生長曲線 86
圖5.12 在SF-MNC中WBC與CD34+ cell長期培養的結果 86
圖5.13 培養前與培養後細胞的端粒酶基因的RT-PCR分析 87
圖5.14 培養前與培養後細胞的端粒酶活性分析 88
圖5.15 CD133+ cell經過SF-HSC七天培養後所分泌的細胞激素 89
圖5.16 MNC經過SF-MNC培養六天後所分泌的細胞激素 90
圖5.17 Scale-up所用的各種不同培養器皿 91

圖5.18 CD133+ cell在24-well plate、FEP Bag、T-25 flask
以及T-75 flask中,以SF-HSC培養七天後的結果 92
圖5.19 MNC在24-well plate、FEP Bag、T-25 flask以及
T-75 flask中,以SF-MNC培養七天後的結果 92

表 目 錄

表2.1 幹細胞移植的案例 13
表2.2 人類與老鼠造血幹細胞的特殊表面抗原 21
表5.1 在IMDM+10% FBS中篩選9種細胞激素的29-5實驗
設計矩陣及結果 93
表5.2在IMDM+10% FBS中篩選4種細胞激素的24-1實驗
設計矩陣及結果 93
表5.3在IMDM+10% FBS中4種細胞激素的陡升路徑及結果 94
表5.4 在IMDM+TISF中篩選8種血清取代物的28-4實驗
設計矩陣及結果 94
表5.5在IMDM+TISF中篩選4種血清取代物的24實驗
設計矩陣及結果 95
表5.6在IMDM+TISF中4種血清取代物的陡升路徑及結果 95
表5.7 在IMDM+BIT2中篩選7種細胞激素的27-3實驗設計
矩陣及結果 96
表5.8在IMDM+BIT2中7種細胞激素的陡升路徑及結果 96
表5.9 BIT2與CC-S6在不同基礎與商業培養基中的比較 97
表5.10 CD133+ cell在SF-HSC與MNC在SF-MNC批次
培養前後的細胞表現 97
表5.11在IMDM+CC-S9中篩選4種血清取代物的24實驗
設計矩陣及結果 98
表5.12在IMDM+CC-S9中3種血清取代物的陡升路徑及結果 98
表5.13在IMDM+BIT中篩選10種細胞激素的210-6實驗
設計矩陣及結果 99
表5.14在IMDM+BIT中9種細胞激素的陡升路徑及結果 99
表5.15 BIT與CC-S9在不同基礎與商業培養基中的比較 100
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