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研究生:邱盈瑛
研究生(外文):Yin-Ying Chiu
論文名稱:以造血先驅細胞體外增殖探討骨髓間質細胞條件培養基之生產系統
論文名稱(外文):The investigation on the bone marrow stromal condition medium production systems for ex vivo expansion
指導教授:邱紫文邱紫文引用關係
指導教授(外文):Tzyy-Wen Chiou
學位類別:碩士
校院名稱:國立東華大學
系所名稱:生物技術研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:82
中文關鍵詞:骨髓間質細胞條件培養基造血先驅細胞體外增殖
外文關鍵詞:bone marrow stromal cellcondition mediumhematopoietic progenitor cellex vivo expansion
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血液先驅細胞移植在臨床上已被肯定並應用於許多致命性疾病的治療。骨髓間質細胞可以分泌許多造血細胞激素,提供一個與活體內造血系統相似的作用環境。本研究中首先評估骨髓間質細胞之生長動力學,測試不同載體材質與骨髓間質細胞之相容性,以懸浮培養系統探討骨髓間質細胞條件培養基生產系統,利用酵素連結免疫分析法,進行細胞激素分析,以建立最適化骨髓間質細胞培養系統之操作模式。其次,本研究以流式細胞儀進一步探討不同間質細胞(包括人類骨髓間質細胞株HS-5與老鼠骨髓間質細胞株AC6.21)其條件培養基對臍帶血造血先驅細胞增殖分化之影響。結果顯示,老鼠間質細胞株AC6.21條件培養基中含有SCF、FLT-3 ligand與IL-6等造血生長因子,以3g/L Cytodex 1為載體進行懸浮培養,可以有效簡化收取間質細胞培養液程序。人類間質細胞株HS-5條件培養基中含有SCF、GM-CSF、FLT-3 ligand、IL-6與IL-8等造血生長因子,其以2D MicroHex為載體進行懸浮培養,可以有效簡化收取間質細胞培養液程序。以Leukemia inhibitory factor (LIF)刺激培養AC6.21間質細胞的條件培養液來培養臍帶血造血先驅細胞,總細胞數隨著培養時間而增加,在第21天最高,增加為4.2倍, CD34+細胞在第14天達到最高,約增為1.5倍;增殖培養之細胞以表現HLA-DR之細胞最多,傾向淋巴系分化。以HS-5 的條件培養基進行造血先驅細胞體外培養,總細胞數在第7天最高,增加為7倍,CD34+細胞與Thy-1+細胞均略增為1.3倍;以懸浮培養系統製備之HS-5條件培養基培養所得之總細胞數在第7天最高,增加近12倍,CD34+細胞與Thy-1+細胞均略增為2.5與2.1倍。增殖培養之細胞以表現CD71之細胞最高,傾向髓系分化。
Umbilical cord blood (UCB) has been considered as an alternative source of primitive hematopoietic stem/progenitor cells to cure several malignant and nonmalignant diseases in clinic. However, the ex vivo expansion of cord blood hematopoietic progenitor cells may be necessary to engraft adult patients. Through secreted extracellular matrices and cytokine environment, bone marrow stromal cells can promote and regulate the self-renewal, differentiation, and proliferation of stem/progenitor cells. This study is designed to prepare different stromal-free condition media sourced from various stromal culture systems and to compare their effects on the ex vivo expansion of cord blood hematopoietic progenitor cells.
The murine bone marrow stromal cell line, AC6.21, is a fibroblast-like attachment cell. In the LIF-treated AC6.21 stromal cell condition media(SCM-LIF), several hematopoietic growth factors, including stem cell factor, interleukin-6 and FLT-3 ligand, were detected. It was found that Cytodex 1, as a carrier in the spinner flask culture system, could support the growth of AC6.21 and then the higher cell density was achieved. In the CB ex vivo expansion experiments, the total cell number was expanded 4.2 fold within 21 days. The AC6.21 SCM-LIF supported hematopoietic progenitor cells differentiating to lymphoid lineage.
The human bone marrow stromal HS-5 cell line is a shear-sensitive fibroblast-like attachment cell, which can secrete several hematopoietic factors such as GM-CSF, SCF, IL-3, IL-6, and IL-8 without additional cytokine stimulation. The specific growth rate was 0.004 hr-1 and the glucose uptake rate was 5.5×10-8 mg/hr per cell in the T-flask culture system. Compared to the T-flask culture system, 2D MicroHex carriers in spinner flask culture system could result in increased cytokine production. In the ex vivo expansion experiments, the HS-5 condition medium supported the differentiation of hematopoietic progenitor cells to myeloid lineage.
誌謝…………………………………………………………………………....I
中文摘要……………………………………………………………………...II
Abstract………………………………………………………………….…. .IV
目錄…………………………………………………………………….....…...V
圖目錄……………………………………………………………………...VIII
表目錄…………………………………………………………………………X
中英名詞對照………………………………………………………………..XI

第一章 緒論…………………………………………………………………..1
1.1 研究背景………………………………………………………………...1
1.2 研究目的………………………………………………………………....2
第二章 文獻回顧…………………………………………………………….3
2.1 造血幹細胞………………………………………………………………3
2.1.1 人類造血幹/先驅細胞移植…………………………………………...3
2.1.2 骨髓移植……………………………………………………………..4
2.1.3 周邊血移植…………………………………………………………..4
2.1.4 臍帶血移植…………………………………………………………..5
2.1.5 臍帶血幹細胞………………………………………………………..8
2.2 血液幹/先驅細胞體外增殖……………………………………………….6
2.2.1造血細胞臨床增殖培養系統………………………………………….7
2.2.2 平面培養系統………………………………………………………..7
2.2.3 攪拌式懸浮培養系統………………………………………………...8
2.2.4 微載體培養系統……………………………………………………..9
2.3 造血生長因子…………………………………………………………..10
2.4 間質細胞………………………………………………………………..14
2.5 無間質細胞培養系統…………………………………………………...16
2.6 反應器系統培養………………………………………………………...17
2.7 人類造血幹細胞活性測試………………………………………………21
第三章 實驗流程與方法…………………………………………………...24
3.1 骨髓間質細胞培養與條件培養基之製備………………………………..25
3.1.1 老鼠骨髓間質細胞株AC 6.21………………………………………25
3.1.1.1維持培養…………………………………………………………..25
3.1.1.2 條件培養基之製備………………………………………………..25
3.1.1.3條件培養基之濃縮………………………………………………...26
3.1.2 人類骨髓間質細胞株HS-5(CRL-11882)…………………………..26
3.1.2.1 維持培養…….……………………………………………………26
3.1.2.2 條件培養基之製備………………………………………………..27
3.2 微載體培養系統……………………………………………………...…27
3.2.1 微載體前處理………………………………………………………27
3.2.2 細胞接種與培養……………………………………………………28
3.3 臍帶血收集……………………………………………………………..28
3.3.1 單核細胞之分離……………………………………………………29
3.3.2 CD34+細胞之篩選與分析…………………………………….…..…29
3.3.3 冷凍保存細胞………………………………………………………30
3.3.4 流式細胞儀…………………………………………………………31
3.4 細胞數之定量………………………………………………………...…31
3.5 代謝分析………………………………………………………………..32
3.5.1葡萄糖分析………………………………………………………….32
3.5.2 乳酸分析……………………………………………………………32
3.6 細胞激素之分析………………………………………………………...32
3.7 幹細胞體外培養條件之探討……………………………………………32
第四章 實驗結果分析與討論………………………………………………34
4.1老鼠骨髓間質細胞株AC 6.21…………………………………………...34
4.1.1 老鼠骨髓間質細胞AC6.21生長動力學之探討……………………..34
4.1.2老鼠骨髓間質細胞AC6.21代謝分析………………………………..37
4.1.3 老鼠間質細胞AC6.21之細胞激素定量分析………………………..39
4.1.4 老鼠間質細胞條件培養基對造血先驅細胞增殖之影響…………….40
4.1.5 懸浮培養系統吸附材質之比較……………………………………..47
4.1.6 間質細胞AC6.21微載體培養系統之探討………………………….50
4.2 人類骨髓間質細胞株HS-5……………………………………………...53
4.2.1 人類骨髓間質細胞株HS-5生長動力學之探討……………………..53
4.2.2 人類骨髓間質細胞株HS-5之代謝分析…………………………….56
4.2.3 懸浮培養系統吸附材質之比較……………………………………..57
4.2.4 人類骨髓間質細胞HS-5微載體培養系統之探討…………………..61
4.2.5 人類骨髓間質細胞HS-5之細胞激素定量分析……………………..63
4.2.6 人類間質細胞條件培養基HS-5 CM對造血先驅細胞增殖之影響….65
4.3 實驗結果討論……………………………………………………………72
第五章 結論…………………………………………………………………74
第六章 參考文獻……………………………………………………………77






圖目錄

圖2-1 流式細胞儀螢光偵測原理示意圖……………………………………23
圖3-1 實驗流程示意圖……………………………………………………..24
圖3-2 微載體懸浮培養系統示意圖…………………………………………28
圖4-1 老鼠間質細胞AC6.21於不同培養基之生長情形……………………35
圖4-2 老鼠間質細胞AC6.21於T-flask之生長情形………………………..36
圖4-3 AC6.21於不同培養基培養期間之葡萄糖濃度變化………………….38
圖4-4 平面培養收集AC6.21條件培養基之細胞激素定量分析…………….39
圖4-5-a 老鼠間質細胞條件培養基對造血細胞增殖之影響…………………..43
圖4-5-b 老鼠間質細胞條件培養基對造血細胞增殖之影響…………………..44
圖4-6-a 流式細胞儀分析以老鼠間質細胞條件培養基體外增殖培養後對造血細胞表面抗原表現之影響….…………………………………………..45
圖4-6-b 流式細胞儀分析以老鼠間質細胞條件培養基體外增殖培養後對造血細胞表面抗原表現之影響.……………………………………………..46
圖4-7 加入AC6.21細胞培養Cytodex 1微載體之之染色觀察情形………...48
圖4-8 加入AC 6.21培養後Cytodex 1之觀察情形…………………………48
圖4-9 加入AC6.21細胞培養後Cytopore之觀察情形………………………49
圖4-10 加入AC6.21細胞培養後CultiSpher-G之觀察情形………………….49
圖4-11 間質細胞AC6.21在Cytodex 1 懸浮培養之生長情形………………..52
圖4-12 人類間質細胞HS-5於平面培養之生長情形…………………………54
圖4-13 人類間質細胞HS-5平面培養之生長動力學…………………………55
圖4-14 間質細胞HS-5於平面培養期間葡萄糖濃度消耗累積量……………..56
圖4-15 加入HS-5培養後Cytodex 1之觀察情形………………………….…58
圖4-16 加入HS-5細胞培養後CultiSpher-G之觀察情形…………………….59
圖4-17 未加入HS-5細胞培養之2D MicroHex微載體………………………60
圖4-18 加入HS-5細胞培養後2D MicroHex之觀察情形……………………60
圖4-19 間質細胞HS-5於2D MicroHex之生長情形…………………………62
圖4-20 人類骨髓間質細胞HS-5於懸浮培養系統之IL-6與GM-CSF累積濃度之變化………….……………………………………………………64
圖4-21 人類骨髓間質細胞HS-5於懸浮培養系統之SCF、IL-3和GM-CSF累積濃度變化……….……………………………………………………64
圖4-22-a 人類間質細胞條件培養基對造血細胞增殖之影響……………….….68
圖4-22-b 人類間質細胞條件培養基對造血細胞增殖之影響…………………...69
圖4-23-a 流式細胞儀分析以人類間質細胞條件培養基體外增殖培養後對造血細胞表面抗原表現之影響……………………………………………...70
圖4-23-b 流式細胞儀分析以人類間質細胞條件培養基體外增殖培養後對造血細胞表面抗原表現之影響………………………………………………………..71

表目錄

表2-1 平面培養與懸浮微載體培養之比表面積…………………………….10
表2-2 以不同細胞激素組合刺激人類造血細胞增殖之培養系統比較表……19
表3-1 收取之臍帶血各項資料……………………………………………...30
表4-1 以平面培養系統收集條件培養基之間質細胞生長變化……...………37
表4-2 AC6.21條件培養基對造血細胞表現之影響…………………………42
表4-3 不同材質微載體之比較……………………………………………...47
表4-4 AC6.21於不同培養系統之生長動力學參數………………….………51
表4-4 不同材質微載體之比較(續)…………………………………………57
表4-5 不同培養系統收集人類骨髓間質細胞條件培養基HS-5 CM之細胞激素分析……..…………………………………………………………...63
表4-6 HS-5條件培養基對造血細胞抗原表現之分析…….…………………67
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