跳到主要內容

臺灣博碩士論文加值系統

(35.174.62.102) 您好!臺灣時間:2021/07/25 04:14
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:葉凌君
研究生(外文):Ling-Chun Yeh
論文名稱:轉錄因子c-Myb作為造血幹細胞標記之可能性
論文名稱(外文):Transcription factor c-Myb as a potential HSC marker
指導教授:呂健惠
指導教授(外文):Chien-Hei Lieu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:英文
中文關鍵詞:造血幹細胞c-Myb
外文關鍵詞:hematopoietic stem cellc-Myb
相關次數:
  • 被引用被引用:0
  • 點閱點閱:173
  • 評分評分:
  • 下載下載:16
  • 收藏至我的研究室書目清單書目收藏:0
本論文利用in situ RT-PCR的方法來偵測特定的轉錄因子(TF),希望能發展出較快速又經濟的方法,來判定細胞處於分化的早期(幹細胞)或是後期。我們先以不同品系、不同時期的血癌細胞株Jurkat (T cell) 、K562 (CML) 及 U-937(promonocyte)細胞建立in situ RT-PCR的方法,再以此法偵測不同的轉錄因子如c-Myb、PU.1、GATA-2 或是幹細胞相關的基因nucleostemin,我們發現在成熟的周邊血白血球細胞中,只有小於1%的細胞會表現c-Myb。接著又利用臍帶血分離出單核細胞並進行CFU assay,我們發現CFU-GEMM, CFU-GM,及BFU-E各有不同程度的c-Myb表現。將in situ RT-PCR的染色強弱分成從0 到4+五個等級,發現這些細胞群落大部份都是3+或2+細胞,但其4+細胞的比率有逐漸從CFU-GEMM的16.1%下降到BFU-E的4%。而自臍帶血分離出CD34+的細胞中,則發現有約40%的細胞表現為4+。由此可知c-Myb的表現明顯的隨著細胞成熟而降低,而在最早期的造血幹細胞時期為最高點。以流式細胞儀與CFU assay鑑定CD34+的細胞中之幹細胞含量,其代表幹細胞的細胞百分比也與c-Myb 4+細胞大致相合。此外,我們還利用血癌細胞株K562,以hemin與TPA誘導其分化成紅血球與血小板,發現在加入100μM hemin培養四天後,c-Myb表現也由00%
4+降低到只有9%的4+ 細胞。另一方面,在加入100 nM TPA培養五天之後,K562其c-Myb表現與未加藥處理的細胞相比,也有明顯減少的傾向。證明在細胞分化成成熟細胞的過程中,C-Myb的表現會由多至少,因此我們相信in situ c-Myb法可以直接以表現的程度區別HSC和progenitor細胞。在臨床須要偵測HSC時,可以作為傳統的CD34 flow cytometry之外另一個指標。
The aim of this study is to set up an in situ RT-PCR method for detecting specifichematopoietic transcription factors (TF) that can be used as stem cell merker. First weused leukemia cell lines including Jurkat, K562, and U-937 cells to set up the in situRT-PCR. Then we found that among GATA-2, PU.1, c-Myb and nucleostemin, onlyc-Myb had less than 1% expression in PBMC. Then we used cord blood mononuclear cells (CBMC)) for CFU assay. CFU-GEMM, CFU-GM, and BFU-E all had c-Mybexpression but in different degree. The expression of c-Myb can be graded from 4+ to0 according to their staining pattern. The percentage of 4+ cells was graduallydecreased from 16.1% in CFU-GEMM to 4% in BFU-E. In the CD34+ cells isolatedfrom CBMC, the percentage of 4+ cells is about 40%. C-Myb expression was obviously decreased with cell maturity, and reached the highest in the CD34+ cells. Analyzing the stem cell content by flow cytometry and CFU assauy, it also showedthat the percentage of stem cell was consistent with the percentage of c-Myb 4+ cells.We also induced K562 to differentiation into erythroid and megakaryocyte by heminand TPA. The c-Myb expression was decreased from 100% 4+ cells in control to 9%
and 35% when treated by hemin and TPA, respectly. Thosedata suggested thatexpression of c-Myb was decrease with cell maturation and in situ c-Myb detection iscapable to distinguish the immature HSC and progenitor cells by simply analyzingtheir expression pattern. We believed this method might contribute to the clinicalHSC detection as an alternate indicator beside the CD34 measurement.
K, Traver D, Miyamoto T, and Weissman I L. A clonogenic
common myeloid progenitor that gives rise to all myeloid lineages.
Nature 404, 193-197 (2000).
‧ Anderson KL, Smith KA, Perkin H, Hermanson G, Anderson CG,
Jolly DJ, Maki RA, Torbett BE. PU.1 and the granulocyte- and
macrophage colony-stimulating factor receptors play distinct roles in
late-stage myeloid cell differentiation. Blood 94, 2310-2318 (1999).
‧ Anderson KL, Smith KA, Pio F, Torbett BE, Maki RA. Neutrophils
deficient in PU.1 do not terminally differentiate or become
functionally competent. Blood 92, 1576-1585 (1998).
‧ Anfossi G, Gewirtz AM, Calabretta B. An oligomer complementary to
c-myb-encoded mRNA inhibits proliferation of human myeloid
leukemia cell lines. Proc. Natl Acad. Sci USA. 86, 3379-3383 (1989).
‧ Barreda DR., Belosevic M. Transcriptional regulation of
hematopoiesis. Dev. Comp. Immunol. 25, 763-789 (2001).
‧ Berenson RJ, Bensinger WI, Hill RS, Andrews RG, Garcia-Lopez J,
Kalamasz DF, Still BJ, Spitzer G, Buckner CD, Bernstein ID.
Engraftment after infusion of CD34+ marrow cells in patients with
breast cancer or neuroblastoma. Blood 77, 1717-1722 (1991).
‧ Bhatia M, Bonnet D, Murdoch B, Gan OI, Dick JE. A newly
discovered class of human hematopoietic cells with
SCID-repopulating activity. Nat. Med. 4, 1038-1045 (1998).
‧ Bjerregaard MD, Jurlander J, Klausen P, Borregaard N, Cowland JB.
The in vivo profile of transcription factors during neutrophil
differentiation in human bone marrow. Blood 101, 4322-4332 (2003).
‧ Boiret N, Rapatel C, Boisgard S, Charrier S, Tchirkov A, Bresson C,
Camilleri L, Berger J, Guillouard L, Guerin JJ, Pigeon P, Chassagne J,
Berger MG. CD34+CDw90(Thy-1)+ subset collocated with
mesenchymal progemitoes in human normal bone marrow hematon
units is enriched in colony-forming unit megakaryocytes and
long-term culture-initiating cells. Exp. Hematol. 31, 1275-1283
(2003).
‧ Cantor AB, Orkin SH. Hematopoietic development: a balancing act.
Curr. Opin. Genet. Dev. 11, 513-519 (2001).
Cross MA, Enver T. The lineage commitment of haemopoietic
progenitor cells. Curr. Opin. Genet. Dev. 7, 609-613 (1997).
‧ DeKoter RP, Lee HJ, Singh H. PU.1 regulates expression of the
interleukin-7 receptor in lymphoid progenitors. Immunity 16, 297-309
(2002).
‧ DeKoter RP, Singh H. Regulation of B lymphocyte and macrophage
development by graded expression of PU.1. Science 288, 1439-41
(2000).
‧ DeKoter RP, Walsh JC, Singh H. PU.1 regulates both
cytokine-dependent proliferation and differentiation of
granulocyte/macrophage progenitors. EMBO J. 17, 4456-4468 (1998).
‧ Doi H, Inaba M, Yamamoto Y, Taketani S, Mori SI, Sugihara A, Ogata
H, Toki J, Hisha H, Inaba K, Sogo S, Adachi M, Matsuda T, Good RA,
Ikehara S. Pluripotent hemopoietic stem cells are c-kitAcad. Sci. U S A 94, 2513-2517 (1997).
‧ Emambokus N, Vegiopoulos A, Harman B, Jenkinson E, Anderson G,
Frampton J. Progression through key stages of hematopoiesis is
dependent on distinct threshold levels of c-Myb. EMBO J. 22,
4478-4488 (2003).
‧ Engelhardt M, Lübbert M, and Guo Y. CD34+ or CD34-: which is the
more primitive? Leukemia 16, 1603-1608 (2002).
‧ Evans T, Felsenfeld G. The erythroid-specific transcription factor
Eryf1: a new finger protein. Cell 58, 877-885 (1989).
‧ Feuring-Buske M, Hogge DE. Hoechst 33342 efflux identifies a
subpopulation of cytogenetically normal CD34(+)CD38(-) progenitor
cells from patients with acute myeloid leukemia. Blood 97, 3882-3889
(2001).
‧ Fujiwara Y, Chang AN, Williams AM, Orkin SH. Functional overlap
of GATA-1 and GATA-2 in primitive hematopoietic development.
Blood 103, 583-585 (2004).
‧ Gewirtz AM, Anfossi G, Venturelli D, Valpreda S, Sims R, Calabretta
B. G1/S transition in normal human T-lymphocytes requires the
nuclear protein encoded by c-myb. Science 245, 180-183 (1989).
‧ Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC. Isolation
and functional properties of murine hematopoietic stem cells that are
replicating in vivo. J. Exp. Med. 183, 1797-1806 (1996).
‧ Graf T. Differentiation plasticity of hematopoietic cells. Blood 99,
3089-4039 (1997).
Haan GD and Ploemacher R. Methods in molecular medicine,
Hematopoietic stem cell protocol. Humana Press. 63, 143-151 (2002).
‧ Haga SB, Fu S, Karp JE, Ross DD, Williams DM, Hankins WD,
Behm F, Ruscetti FW, Chang M, Smith BD, Becton D, Raimondi SC,
Berg PE. BP1, a new homeobox gene, is frequently expressed in acute
leukemias. Leukemia 11, 1867-1875 (2000).
‧ Harigae H, Takahashi S, Suwabe N, Ohtsu H, Gu L, Yang Z, Tsai FY,
Kitamura Y, Engel JD, Yamamoto M. Differential roles of GATA-1
and GATA-2 in growth and differentiation of mast cells. Genes Cells 3,
39-50 (1998).
‧ Hernandez-Munain C, Krangel MS. Regulation of the T-cell receptor
delta enhancer by functional cooperation between c-Myb and
core-binding factors. Mol. Cell Biol. 14, 473-483 (1994).
‧ Ikonomi P, Noguchi CT, Miller W, Kassahun H, Hardison R,
Schechter AN. Levels of GATA-1/GATA-2 transcription factors
modulate expression of embryonic and fetal hemoglobins. Gene 261,
277-287 (2000).
‧ Katherine A. Staskus, Janet E. Embretson, Ernest F. Retzel, Janet
Beneke and Ashley T. Haase. PCR IN SITU: New technologies with
single cell resolution for the detection and investigation of viral
lantency and persistence. The Regents of the University of Minnesota.
(1994)
‧ Kazuhiro O, Ko S, Tahir T. Eukaryotic transcriptional regulatory
complexes: cooperativity from near and afar. Curr. Opin. Struc. Biol.
13, 40-48 (2003).
‧ Kim M, Morshead CM. Distinct populations of forebrain neural stem
and progenitor cells can be isolated using side-population analysis. J
Neurosci. 23, 10703-10709 (2003).
‧ Kumar A, Lee CM, Reddy EP. c-Myc is essential but not sufficient for
c-Myb-mediated block of granulocytic differentiation. J. Biol. Chem.
278, 11480-11488 (2003).
‧ Labbaye C, Valtieri M, Barberi T, Meccia E, Masella B, Pelosi E,
Condorelli GL, Testa U, Peschle C. Differential expression and
functional role of GATA-2, NF-E2, and GATA-1 in normal adult
hematopoiesis. J. Clin. Invest. 95, 2346-2358 (1995).
‧ Lin HH, Sternfeld DC, Shinpock SG, Popp RA, Mucenski ML.
Functional analysis of the c-myb proto-oncogene. Curr. Top Microbiol.
Immunol. 211, 79-87 (1996).
Litzow MR, Brashem-Stein C, Andrews RG, Bernstein ID.
Proliferative responses to interleukin-3 and granulocyte
colony-stimulating factor distinguish a minor subpopulation of
CD34-positive marrow progenitors that do not express CD33 and a
novel antigen, 7B9. Blood 77, 2354-2359 (1991).
‧ McCilloch EA. Methods in molecular medicine, Hematopoietic stem
cell protocol. Humana Press. 63, 153-160 (2002).
‧ Melchers F, Rolink A. Hematopoietic stem cells: Lympoiesis and the
pattern of commitment versus plasticity. Stem cell biology 307-328
(2001).
‧ Miller CL. and Eaves CL. Methods in molecular medicine,
Hematopoietic stem cell protocol. Humana Press. 63, 123-141 (2002).
‧ Morel F, Galy A, Chen B, Szilvassy SI. Equal distribution of
competitive long-term repopulating stem cells in the CD34+ and
CD34- fractions of Thy-1lowSca-1+ bone marrow cells. Exp. Hematol.
26, 440-448 (1998).
‧ Nakamaki T, Okabe-Kado J, Yamamoto-Yamaguchi Y, Hino K,
Tomoyasu S, Honma Y, Kasukabe T. Role of
MmTRA1b/phosphorlipid scramblase1 gene expression in the
differentiation of human myeloid leukemia cell into granulocyte. Exp.
Hematol. 30, 421-429 (2002).
‧ Nerlov C, Querfurth E, Kulessa H, Graf T. GATA-1 interacts with the
myeloid PU.1 transcription factor and represses PU.1-dependent
transcription. Blood 95, 2543-2551 (2000).
‧ Ness SA. Myb protein specificity: evidence of a context-specific
transcription factor code. Blood Cells Mol. Dis. 31, 192-200 (2003).
‧ Ness SA. Myb protein specificity: evidence of a context-specific
transcription factor code. Blood cell, Mol. Dis. 31, 192-200 (2003).
‧ Oelgeschlager M, Nuchprayoon I, Luscher B, Friedman AD. C/EBP,
c-Myb, and PU.1 cooperate to regulate the neutrophil elastase
promoter. Mol. Cell Biol. 16, 4717-4725 (1996).
‧ Oh IH, Reddy EP. The myb gene family in cell growth, differentiation
and apoptosis. Oncogene 18, 3017-3033 (1999).
‧ Ohneda K, Yamamoto M. Roles of hematopoietic transcription factors
GATA-1 and GATA-2 in the development of red blood cell lineage.
Acta. Haematol. 108, 237-245 (2002).
‧ Orkin SH. GATA-binding transcription factors in hematopoietic cells.
Blood 80, 575-581 (1992).
Tenen Daniel G, Hromas Robbert, Licht Jonathan D., and Zhang
Dong-Er. Transcription factors, Normal myeloid development and
leukemia. Blood 90, 489-519 (1997).
‧ Ting CN, Olson MC, Barton KP, Leiden JM. Transcription factor
GATA-3 is required for development of the T-cell lineage. Nature 384,
474-478 (1996).
‧ Tsai FY, Keller G, Kuo FC, Weiss M, Chen J, Rosenblatt M, Alt FW,
Orkin SH. An early haematopoietic defect in mice lacking the
transcription factor GATA-2. Nature 371, 221-226 (1994).
‧ Tsai FY, Orkin SH. Transcription factor GATA-2 is required for
proliferation/survival of early hematopoietic cells and mast cell
formation, but not for erythroid and myeloid terminal differentiation.
Blood 89, 3636-3643 (1997).
‧ Tsai R Y, McKay RD. A nucleolar mechanism controlling cell
proliferation in stem cells and cancer cells. Genes Dev. 16, 2991-3003
(2002).
‧ Tsai SF, Martin DI, Zon LI, D''Andrea AD, Wong GG, Orkin SH.
Cloning of cDNA for the major DNA-binding protein of the erythroid
lineage through expression in mammalian cells. Nature 339, 446-51
(1989).
‧ Weiss MJ, Orkin SH. GATA transcription factors: key regulators of
hematopoiesis. Exp. Hematol. 23, 99-107 (1995).
‧ Yamaguchi Y, Ackerman SJ, Minegishi N, Takiguchi M, Yamamoto M,
Suda T. Mechanisms of transcription in eosinophils: GATA-1, but not
GATA-2, transactivates the promoter of the eosinophil granule major
basic protein gene. Blood 91, 3447-3458 (1998).
‧ Zanjani ED, Almeida-Porada G, Livingston AG, Flake AW, Ogawa M.
Human bone marrow CD34- cells engraft in vivo and undergo
multilineage expression that includes giving rise to CD34+ cells. Exp.
Hematol. 26, 353-360 (1998).
‧ Zhang DE, Zhang P, Wang ND, et al. Absence of granulocyte
colony-stimulating factor signaling and neutrophil development in
CCFAAT enhancer binding protein alpha-deficient mice. Proc. Natl
Acad. Sci. USA. 94, 569-574 (1997).
‧ Zhang P, Behre G, Pan J, Iwama A, Wara-Aswapati N, Radomska HS,
Auron PE, Tenen DG, Sun Z. Negative cross-talk between
hematopoietic regulators: GATA proteins repress PU.1. Proc. Natl
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關論文