以系統生物學策略探討臍帶血來源之造血幹細胞分子調控網路

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姓名

劉采琪(Tsai-Chi Liu) 查詢紙本館藏

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系統生物與生物資訊研究所

論文名稱

以系統生物學策略探討臍帶血來源之造血幹細胞分子調控網路
(Systems Biology Approaches to Explore Molecular Network in Human Cord Blood-Derived Hematopoietic Stem Cells)

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摘要(中)

迄今為止，臍帶血來源的造血幹細胞體外擴增培養技術已趨成熟，克服了幹細胞在臨床移植上數量不足的問題。然而，至今尚不清楚在不同體外培養系統下產生的這些造血幹細胞基因組功能差異。本研究的目的為使用有血清及無血清兩種不同體外擴增培養方式的造血幹細胞，分析兩者有差異表現的基因，探討分子功能層面上的關係。實驗首先將體外擴增培養的造血幹細胞進行RNA提取，接著進行微陣列基因表達晶片分析，篩選出在有血清及無血清培養系統下有差異表達的基因，總共有839個基因，再針對這些基因進行功能描述分析，結果顯示大部分的基因為細胞激素及趨化因子，富集在TNF信號通路，調控血清擴增培養的造血幹細胞發炎、免疫反應。此外，我們進一步使用qPCR分析了調控造血幹細胞功能重要的基因CCL2（C-C motif chemokine ligand 2）、TNF（tumor necrosis factor）和FOS（FBJ murine osteosarcoma viral oncogene homolog）。實驗數據顯示使用體外擴增血清培養系統的造血幹細胞將誘導炎症反應和CD38的高表達，我們認為是胎牛血清內含的異種物質可能刺激造血幹細胞在體外擴增的過程中引起發炎表徵並誘導癌症標誌物CD38表達。本研究提供了造血幹細胞在不同體外擴增培養環境下的廣泛轉錄譜，對造血幹細胞的分子調控網路提供新的見解。

摘要(英)

To date, different experimental strategies have been developed for the ex vivo expansion of human hematopoietic stem (HSCs) in clinical application. However, it is still unclear to what difference in genomic function in HSCs expansion under different culture systems. In this study, we compared gene-expression profile of ex vivo expanded serum (10% FBS, fetal bovine serum) and serum-free culture systems, and then analyze molecular function of differentially expressed gene using microarray chips. We identified 839 differentially expressed genes between two culture systems. These genes were enriched in TNF regulated inflammatory pathway in FBS culture system. In addition, mRNA expression of CCL2 (C-C motif chemokine ligand 2) 、 TNF (tumor necrosis factor) and FOS (FBJ murine osteosarcoma viral oncogene homolog) was validated by RT-qPCR. Our data revealed that ex vivo expansion of HSCs using FBS culture system would induce the inflammation response and high expression of CD38. It indicated that FBS culture system might cause inflammation pathway and induce the cancer marker CD38 expression during ex vivo expansion of HSCs. This study provided the transcriptional profile, and new insights into the genomic functionality of HSCs under different expanded cultures.

關鍵字(中)

★ 新生兒臍帶血
★ 造血幹細胞
★ 體外擴增培養系統
★ 系統生物學策略
★ 基因表達譜
★ 分子網路

關鍵字(英)

★ Human cord blood
★ Hematopoietic stem cells
★ Ex vivo expansion
★ Systems biology approaches
★ Gene expression profiles
★ Molecular network

論文目次

目錄
中文摘要 ii
Abstract iii
目錄 iv
圖目錄 vii
表目錄 viii
一、緒論 1
1-1 造血幹細胞 1
1-2 造血幹細胞鑑定 2
1-3 造血幹細胞擴增 4
1-3-1 血清擴增培養 6
1-3-2 無血清擴增培養 6
1-4 造血幹細胞的研究及臨床應用 7
1-5 DNA微陣列分析 8
1-6 KEGG資料庫 9
1-7 WIkiPathways資料庫 10
1-8 GO資料庫 10
1-9 Cytoscape資料庫 10
研究動機與目的 11
二、實驗材料與方法 12
2-1 實驗材料 12
2-1-1 細胞培養 12
2-1-2 造血幹細胞表面抗原分析材料 13
2-1-3 微陣列晶片及分析軟體 13
2-1-4 RNA萃取套件及RT-qPCR反應試劑 14
2-1-5 蛋白質萃取套件及西方墨點法反應試劑 14
2-2 實驗方法 15
2-2-1 源於臍帶血中的造血幹細胞分離、純化 15
2-2-2 造血幹細胞體外擴增培養系統 15
2-2-3 細胞表面抗原分析 16
2-2-4 RNA萃取 16
2-2-5 DNA微陣列晶片實驗 17
2-2-6 微陣列數據分析 18
2-2-7 功能性註解和生物途徑富集分析 18
2-2-8 qPCR（即時定量聚核酶反應） 19
2-2-9 蛋白質萃取及定量 22
2-2-10 西方墨點法 22
2-2-11 ELISA (酵素免疫分析法) 23
三、實驗流程 25
四、實驗結果 27
4-1 體外擴增培養的造血幹細胞 27
4-1-1 血清擴增培養下的HSCs CD38表達增加 (流式細胞儀分析圖由元智大學姚少凌教授提供) 27
4-1-2 其他造血幹細胞表面標記的表達 29
4-2 在不同培養系統下HSCs的全基因表達譜 30
4-3 為差異表現基因做功能性註解 33
4-4 利用qRT-PCR以及西方墨點法來應證DNA微陣列分析的結果 42
4-5 藉ELISA實驗與流式細胞技術佐證HSCs內含CCL2高表現（流式細胞儀分析圖由元智大學姚少凌教授提供） 44
4-6 來自其他成熟血細胞的CD marker表現 46
五、討論與結論 48
5-1 探討14條overlapping pathway調控造血幹細胞的機制 48
5-2 血清擴增培養系統的造血幹細胞更具有免疫、發炎的功能 50
5-3 血清因子對造血幹細胞的影響 50
5-4 CCL2促進血清擴增培養的造血幹細胞分化 54
5-5 CD38誘導血清擴增培養下的造血幹細胞具癌幹細胞的特性 54
5-6 結論 55
5-7 未來展望 55
六、參考文獻 57
七、附錄表 62

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指導教授

許藝瓊(Yi-Chiung Hsu)

審核日期

2019-7-25

推文