博碩士論文 106826002 詳細資訊




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姓名 劉采琪(Tsai-Chi Liu)  查詢紙本館藏   畢業系所 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
參考文獻 參考文獻
1. Jagannathan-Bogdan M, Zon LI: Hematopoiesis. Development (Cambridge, England) 2013, 140(12):2463-2467.
2. Department of Health and Human Services. 5. Hematopoietic Stem Cells. June 2001 < Available at: https://stemcells.nih.gov/info/2001report/chapter5.htm>.
3. Yuk Yin Ng MRMB, Edwin F.E. de Haas, Karin Pike-Overzet, and Frank J.T. Staal: Isolation of Human and Mouse Hematopoietic Stem Cells, vol. 506; 2009.
4. GERALD J. SPANGRUDE SH, IRVING L. WEISSMAN: Purffication and Characterization of Mouse Hematopoietic Stem Cells. SCIENCE 1988, 24I(4861):58-62.
5. Sidney LE, Branch MJ, Dunphy SE, Dua HS, Hopkinson A: Concise review: evidence for CD34 as a common marker for diverse progenitors. Stem cells (Dayton, Ohio) 2014, 32(6):1380-1389.
6. Yin AH, Miraglia S, Zanjani ED, Almeida-Porada G, Ogawa M, Leary AG, Olweus J, Kearney J, Buck DW: AC133, a novel marker for human hematopoietic stem and progenitor cells. Blood 1997, 90(12):5002-5012.
7. Handgretinger R, Kuci S: CD133-Positive Hematopoietic Stem Cells: From Biology to Medicine. Advances in experimental medicine and biology 2013, 777:99-111.
8. Takahashi M, Matsuoka Y, Sumide K, Nakatsuka R, Fujioka T, Kohno H, Sasaki Y, Matsui K, Asano H, Kaneko K et al: CD133 is a positive marker for a distinct class of primitive human cord blood-derived CD34-negative hematopoietic stem cells. Leukemia 2014, 28(6):1308-1315.
9. Mainou-Fowler T, Dignum HM, Proctor SJ, Summerfield GP: The prognostic value of CD38 expression and its quantification in B cell chronic lymphocytic leukemia (B-CLL). Leukemia & lymphoma 2004, 45(3):455-462.
10. Matrai Z: CD38 as a prognostic marker in CLL. Hematology (Amsterdam, Netherlands) 2005, 10(1):39-46.
11. Ballen KK, Gluckman E, Broxmeyer HE: Umbilical cord blood transplantation: the first 25 years and beyond. Blood 2013, 122(4):491-498.
12. Henig I, Zuckerman T: Hematopoietic stem cell transplantation-50 years of evolution and future perspectives. Rambam Maimonides medical journal 2014, 5(4):e0028.
13. Hofmeister CC, Zhang J, Knight KL, Le P, Stiff PJ: Ex vivo expansion of umbilical cord blood stem cells for transplantation: growing knowledge from the hematopoietic niche. Bone marrow transplantation 2007, 39(1):11-23.
14. Dahlberg A, Delaney C, Bernstein ID: Ex vivo expansion of human hematopoietic stem and progenitor cells. Blood 2011, 117(23):6083-6090.
15. Jing D, Fonseca AV, Alakel N, Fierro FA, Muller K, Bornhauser M, Ehninger G, Corbeil D, Ordemann R: Hematopoietic stem cells in co-culture with mesenchymal stromal cells--modeling the niche compartments in vitro. Haematologica 2010, 95(4):542-550.
16. Schoemans H, Theunissen K, Maertens J, Boogaerts M, Verfaillie C, Wagner J: Adult umbilical cord blood transplantation: a comprehensive review. Bone marrow transplantation 2006, 38(2):83-93.
17. Mousavi SH, Abroun S, Soleimani M, Mowla SJ: Expansion of human cord blood hematopoietic stem/progenitor cells in three-dimensional Nanoscaffold coated with Fibronectin. International journal of hematology-oncology and stem cell research 2015, 9(2):72-79.
18. Albeniz I, Turker-Sener L, Bas A, Kalelioglu I, Nurten R: Isolation of hematopoietic stem cells and the effect of CD38 expression during the early erythroid progenitor cell development process. Oncol Lett 2012, 3(1):55-60.
19. Yao CL, Chu IM, Hsieh TB, Hwang SM: A systematic strategy to optimize ex vivo expansion medium for human hematopoietic stem cells derived from umbilical cord blood mononuclear cells. Experimental hematology 2004, 32(8):720-727.
20. Madlambayan GJ, Rogers I, Kirouac DC, Yamanaka N, Mazurier F, Doedens M, Casper RF, Dick JE, Zandstra PW: Dynamic changes in cellular and microenvironmental composition can be controlled to elicit in vitro human hematopoietic stem cell expansion. Experimental hematology 2005, 33(10):1229-1239.
21. Kita K, Xiu F, G Jeschke M: Ex vivo expansion of hematopoietic stem and progenitor cells: Recent advances. World Journal of Hematology 2014, 3:18.
22. REKERS PE, COULTER MP, WARREN SL: EFFECT OF TRANSPLANTATION OF BONE MARROW INTO IRRADIATED ANIMALS. JAMA Surgery 1950, 60(4):635-667.
23. Hatzimichael E, Tuthill M: Hematopoietic stem cell transplantation. Stem Cells Cloning 2010, 3:105-117.
24. Southern EM: Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of molecular biology 1975, 98(3):503-517.
25. Govindarajan R, Duraiyan J, Kaliyappan K, Palanisamy M: Microarray and its applications. Journal of pharmacy & bioallied sciences 2012, 4(Suppl 2):S310-312.
26. Kanehisa M, Goto S, Hattori M, Aoki-Kinoshita KF, Itoh M, Kawashima S, Katayama T, Araki M, Hirakawa M: From genomics to chemical genomics: new developments in KEGG. Nucleic acids research 2006, 34(Database issue):D354-357.
27. Kanehisa M, Furumichi M, Tanabe M, Sato Y, Morishima K: KEGG: new perspectives on genomes, pathways, diseases and drugs. Nucleic acids research 2016, 45(D1):D353-D361.
28. Kelder T, van Iersel MP, Hanspers K, Kutmon M, Conklin BR, Evelo CT, Pico AR: WikiPathways: building research communities on biological pathways. Nucleic acids research 2012, 40(Database issue):D1301-1307.
29. Kutmon M, Riutta A, Nunes N, Hanspers K, Willighagen EL, Bohler A, Mélius J, Waagmeester A, Sinha SR, Miller R et al: WikiPathways: capturing the full diversity of pathway knowledge. Nucleic acids research 2016, 44(D1):D488-494.
30. Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N, Schwikowski B, Ideker T: Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome research 2003, 13(11):2498-2504.
31. Dahlquist KD, Salomonis N, Vranizan K, Lawlor SC, Conklin BR: GenMAPP, a new tool for viewing and analyzing microarray data on biological pathways. Nature Genetics 2002, 31(1):19-20.
32. Kutmon M, van Iersel MP, Bohler A, Kelder T, Nunes N, Pico AR, Evelo CT: PathVisio 3: an extendable pathway analysis toolbox. PLoS computational biology 2015, 11(2):e1004085.
33. Harris MA, Clark J, Ireland A, Lomax J, Ashburner M, Foulger R, Eilbeck K, Lewis S, Marshall B, Mungall C et al: The Gene Ontology (GO) database and informatics resource. Nucleic acids research 2004, 32(Database issue):D258-261.
34. Yao C-L, Liu C-H, Chu IM, Hsieh T-B, Hwang S-M: Factorial designs combined with the steepest ascent method to optimize serum-free media for ex vivo expansion of human hematopoietic progenitor cells, vol. 33; 2003.
35. Yao CL, Feng YH, Lin XZ, Chu IM, Hsieh TB, Hwang SM: Characterization of serum-free ex vivo-expanded hematopoietic stem cells derived from human umbilical cord blood CD133(+) cells. Stem cells and development 2006, 15(1):70-78.
36. Majeti R, Park CY, Weissman IL: Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood. Cell stem cell 2007, 1(6):635-645.
37. Lapidot T, Kollet O: The essential roles of the chemokine SDF-1 and its receptor CXCR4 in human stem cell homing and repopulation of transplanted immune-deficient NOD/SCID and NOD/SCID/B2mnull mice. Leukemia 2002, 16(10):1992-2003.
38. Kawashima I, Zanjani ED, Almaida-Porada G, Flake AW, Zeng H, Ogawa M: CD34+ human marrow cells that express low levels of Kit protein are enriched for long-term marrow-engrafting cells. Blood 1996, 87(10):4136-4142.
39. Fischer K, Agrawal D: Hematopoietic Stem and Progenitor Cells in Inflammation and Allergy. Frontiers in Immunology 2013, 4(428).
40. Landmann R, Muller B, Zimmerli W: CD14, new aspects of ligand and signal diversity. Microbes and infection 2000, 2(3):295-304.
41. Bulfone-Paus S, Bahri R: Mast Cells as Regulators of T Cell Responses. Frontiers in immunology 2015, 6:394-394.
42. Schuettpelz LG, Link DC: Regulation of hematopoietic stem cell activity by inflammation. Front Immunol 2013, 4:204.
43. Chen G, Goeddel DV: TNF-R1 signaling: a beautiful pathway. Science 2002, 296(5573):1634-1635.
44. Turner MD, Nedjai B, Hurst T, Pennington DJ: Cytokines and chemokines: At the crossroads of cell signalling and inflammatory disease. Biochimica et biophysica acta 2014, 1843(11):2563-2582.
45. Baldridge MT, King KY, Goodell MA: Inflammatory signals regulate hematopoietic stem cells. Trends in immunology 2011, 32(2):57-65.
46. Monlish DA, Bhatt ST, Schuettpelz LG: The Role of Toll-Like Receptors in Hematopoietic Malignancies. Front Immunol 2016, 7:390.
47. Liu T, Zhang L, Joo D, Sun S-C: NF-κB signaling in inflammation. Signal Transduction And Targeted Therapy 2017, 2:17023.
48. Xia Y, Shen S, Verma IM: NF-κB, an active player in human cancers. Cancer immunology research 2014, 2(9):823-830.
49. Taniguchi K, Karin M: NF-κB, inflammation, immunity and cancer: coming of age. Nature Reviews Immunology 2018, 18:309.
50. Kim YK, Shin JS, Nahm MH: NOD-Like Receptors in Infection, Immunity, and Diseases. Yonsei medical journal 2016, 57(1):5-14.
51. Mussano F, Genova T, Corsalini M, Schierano G, Pettini F, Di Venere D, Carossa S: Cytokine, Chemokine, and Growth Factor Profile Characterization of Undifferentiated and Osteoinduced Human Adipose-Derived Stem Cells. Stem cells international 2017, 2017:6202783.
52. Ray A, Joshi J: Cytokines and their Role in Health and Disease: A Brief Overview, vol. 4; 2016.
53. Arango Duque G, Descoteaux A: Macrophage cytokines: involvement in immunity and infectious diseases. Front Immunol 2014, 5:491.
54. DiCarlo J, Agarwal-Hashmi R, Shah A, Kim P, Craveiro L, Killen R, Rosenberg-Hasson Y, Maecker H: Cytokine and chemokine patterns across 100 days after hematopoietic stem cell transplantation in children. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation 2014, 20(3):361-369.
55. Deshmane SL, Kremlev S, Amini S, Sawaya BE: Monocyte chemoattractant protein-1 (MCP-1): an overview. Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research 2009, 29(6):313-326.
56. Marra F, Tacke F: Roles for chemokines in liver disease. Gastroenterology 2014, 147(3):577-594.e571.
57. Zhang J, Lu Y, Pienta KJ: Multiple roles of chemokine (C-C motif) ligand 2 in promoting prostate cancer growth. Journal of the National Cancer Institute 2010, 102(8):522-528.
58. Parameswaran N, Patial S: Tumor necrosis factor-α signaling in macrophages. Critical reviews in eukaryotic gene expression 2010, 20(2):87-103.
59. Landskron G, De la Fuente M, Thuwajit P, Thuwajit C, Hermoso MA: Chronic inflammation and cytokines in the tumor microenvironment. Journal of immunology research 2014, 2014:149185.
60. Chen YJ, Chang LS: Hydroquinone-induced miR-122 down-regulation elicits ADAM17 up-regulation, leading to increased soluble TNF-alpha production in human leukemia cells with expressed Bcr/Abl. Biochemical pharmacology 2013, 86(5):620-631.
61. Shaulian E, Karin M: AP-1 as a regulator of cell life and death. Nature Cell Biology 2002, 4(5):E131-E136.
62. Boyce BF, Yamashita T, Yao Z, Zhang Q, Li F, Xing L: Roles for NF-kappaB and c-Fos in osteoclasts. Journal of bone and mineral metabolism 2005, 23 Suppl:11-15.
63. Trop-Steinberg S, Azar Y: AP-1 Expression and its Clinical Relevance in Immune Disorders and Cancer. The American journal of the medical sciences 2017, 353(5):474-483.
64. Wilkinson AC, Ishida R, Kikuchi M, Sudo K, Morita M, Crisostomo RV, Yamamoto R, Loh KM, Nakamura Y, Watanabe M et al: Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation. Nature 2019, 571(7763):117-121.
65. Collington SJ, Hallgren J, Pease JE, Jones TG, Rollins BJ, Westwick J, Austen KF, Williams TJ, Gurish MF, Weller CL: The role of the CCL2/CCR2 axis in mouse mast cell migration in vitro and in vivo. Journal of immunology (Baltimore, Md : 1950) 2010, 184(11):6114-6123.
66. van de Donk N, Richardson PG, Malavasi F: CD38 antibodies in multiple myeloma: back to the future. Blood 2018, 131(1):13-29.
指導教授 許藝瓊(Yi-Chiung Hsu) 審核日期 2019-7-25
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