臺灣博碩士論文加值系統

血小板對於凝血功能相當重要，而巨核細胞是血小板的前驅細胞。當骨髓中的血球幹細胞開始分化成巨核細胞時，此時血球幹細胞需要接受到血小板生成素 (thrombopoietin, TPO) 與細胞表面上的受體 c-mpl 結合後，透過一連串訊息傳遞刺激血球幹細胞增生以及分化成巨核細胞。在此研究的第一個部分，我們在實驗室建立利用胚胎幹細胞與老鼠的骨髓細胞 (OP9 stromal cell) 共同培養後，在體外將胚胎幹細胞分化成巨核細胞。然而，由於OP9 細胞是直接從老鼠的骨髓取出的初代細胞，其細胞的壽命不長，無法長時間繼代培養。於是我們利用將人類乳突病毒的基因E6/E7藉由反轉錄病毒的載體送入OP9細胞，使得細胞不朽化 (immortalized)， 命名為I-OP9細胞。 相較於正常的OP9細胞，I-OP9細胞可以繼代培養超過四十二代，並且仍然保有其誘發胚胎幹細胞分化成巨核細胞的能力。在此研究的第二部分，我們想了解炭疽桿菌 (Bacillus anthracis) 致死毒素 (lethal toxin, LT) 對巨核細胞的致病影響。炭疽桿菌的主要毒力因子是致死毒素。我們之前的研究已知，致死毒素抑制血小板的功能與致死毒素造成的死亡有關，並且觀察到透過施打致死毒素所造成的死亡前，其小鼠有血小板低下的現象產生。目前已知致死毒素會去抑制訊息傳遞路徑mitogen activated protein kinase (MAPK) 下游的mitogen activated protein kinase kinase (MEK) 的活性，而另外先前已有報導指出巨核細胞的分化與MEK 家族中的extracellular signal-regulated kinase (ERK) 有關；綜合以上事實，我們假設致死毒素抑制血小板的前驅細胞的生成進而造成血小板低下症狀產生。我們利用人類的白血病細胞株 (HEL)、臍帶血單核球細胞、臍帶血幹細胞 (CD34+ cells) 以及老鼠骨髓中的單核球細胞進行體外巨核細胞分化實驗，我們觀察到致死毒素是透過殺死巨核細胞或抑制巨核細胞的分化來抑制血小板生成，而造成血小板生成低下。此外，我們藉由前注射血小板生成素來提升血小板的生成並且觀察到致死毒素所造成的小鼠死亡有明顯的減少。從我們的實驗結果可以得知，致死毒素所造成的致病機轉與其抑制血小板生成有關。

Megakaryocytes are the precursor cells of platelets and critical for maintaining coagulant functions. Thrombopoietin (TPO), the ligand for c-mpl, stimulates proliferation of committed megakaryocytic progenitors and induces maturation of megakaryocytes. In the first part of this study, we established the in vitro megakaryocyte differentiation system from embryonic stem cells co-cultured with OP9 stromal cells. OP9 stromal cells, derived from mouse bone marrow, are useful feeder cells for hematopoiesis in ES cell co-culture systems. However, OP9 is a primary cell line that has a short in vitro life span. Herein, we immortalized primary OP9 cells with the E6 and E7 genes from human papillomavirus type 16. The immortalized OP9 stromal cells were able to extend their lifespan beyond 42 passages and retain their ability to induce megakaryocyte differentiation from embryonic stem cells. In the second part of this study we examined the pathogenic effect of Bacillus anthracis lethal toxin (LT) on megakaryopoiesis. Anthrax LT is the major virulence factor of B. anthracis. In our previous study, we investigated the suppression of platelet function that was associated with LT-induced mortality and observed that LT injection reduces platelet counts prior to death in mice. However, the mechanism responsible for this effect remains unclear. LT is known to inactivate cellular-mitogen-activated protein kinase (MEK) pathways. Previous studies have also shown that the MEK1/2-extracellular signal-regulated kinase (ERK) pathway is critical for megakaryocytic differentiation. Therefore, we hypothesize that LT might inhibit the progenitor cells of platelets, and thereby induce thrombocytopenic responses. We employed the human eruthroleukemia (HEL) cell line, human cord blood-derived mononuclear cells, CD34+ cells,and mouse bone marrow mononuclear cells to perform in vitro megakaryocytic differentiation. These results show that LT suppresses megakaryopoiesis by killing the megakaryocytes and inhibiting megakaryocytic differentiation. Pretreatments with TPO to up-regulate megakaryopoiesis considerably reduced LT-mediated mortality in mice. Our data indicate that LT-suppressed megakaryopoiesis is involved in LT-mediated pathogenesis.

中文摘要 ........................................................................................ I
Abstract ...................................................................................... III
Contents ...................................................................................... V
Introduction .................................................................................. 1
Megakaryopoiesis .............................................................................. 1
Megakaryocyte development ..................................................................... 1
TPO signaling in megakaryopoiesis ............................................................. 2
Megakaryocytic in vitro differentiation ....................................................... 3
Embryonic stem cell ........................................................................... 4
Megakaryocytes differentiated from ES cells ................................................... 5
Megakaryocytes differentiated from cord blood cells ........................................... 5
Megakaryocytes differentiated from bone marrow ................................................ 6
Megakaryocytes differentiated from cell lines ................................................. 7
Anthrax ....................................................................................... 7
The routes of infection of Bacillus anthracis ................................................. 8
The virulence factors of Bacillus anthracis ................................................... 8
How the anthrax toxins interfere with the host signaling pathway .............................. 9
The target cells of anthrax lethal toxin....................................................... 10
Aims of this study ............................................................................ 10
Materials and methods ......................................................................... 13
Cell culture .................................................................................. 13
Preparation of primary MEFs ................................................................... 14
Retroviral vector transduction ................................................................ 15
Tumorigenesis Assay ........................................................................... 15
Megakaryocytic differentiation of ES cells co-cultured with OP9/I-OP9 ......................... 16
RNA isolation and RT-PCR ...................................................................... 17
Acetylcholinesterase staining ................................................................. 17
Immunofluorescence staining ................................................................... 18
Cytotoxicity assay ............................................................................ 18
TPA-induced megakaryocytic differentiation in HEL cells ....................................... 19
Bromodeoxyuridine incorporation assay ......................................................... 19
CFU-MK assay .................................................................................. 20
Preparation of human umbilical mesenchymal stem cells ..........................................20
Megakaryocytic differentiation from cord blood hematopoietic stem cells ....................... 21
Flow cytometric analysis .......................................................................22
DNA content analysis ...........................................................................23
Platelet isolation and activation analysis .................................................... 23
Megakaryocytic differentiation from mouse bone marrow ......................................... 24
Western blotting .............................................................................. 24
TPO treatments on the reduction of LT-induced mortality ....................................... 25
DNA content analysis of megakaryocytes in bone marrow ......................................... 26
Statistics .................................................................................... 27
Results ....................................................................................... 28
Establishment of I-OP9 cells by transducing retroviral vector LXSN16E6E7 ...................... 28
I-OP9 cells increase life span and are tumorigenic ............................................ 29
Characterization of megakaryocytic differentiation from ES cells-morphology examination ................................................................................... 30
Characterization of megakaryocytic differentiation from ES cells-specific markers expression and AchE staining ...................................................................................... 31
Lethal toxin and its cytotoxic activity ....................................................... 32
Effect of LT on megakaryocytic differentiation in HEL cells ................................... 32
LT challenges inhibit human megakaryocyte colony formation .................................... 34
Death of premature/mature megakaryocytes is involved in LT-mediated suppression in a cord-blood-derived CD34+ cell model .................................................................................... 35
LT leads to cell death of megakaryocytes in a model using mouse bone marrow mononuclear cells ......................................................................................... 37
Thrombopoietin treatments reduce LT-mediated mortality, thrombocytopenia, and megakaryopoiesis suppression in mice .......................................................................................... 38
Discussion .................................................................................... 41
Future work ................................................................................... 47
Table 1. Primers used for PCR ................................................................. 48
Figure 1. Detection of E6/E7 mRNA expression in OP9 stromal cells after gene transfer through LXSN16E6E7 retroviral vector. ............................................................................ 49
Figure 2. Proliferative ability of I-OP9 and normal OP9 cells. ................................ 50
Figure 3. Tumorigenecity of I-OP9 cells. ...................................................... 51
Figure 4. Three stages of megakartocytic differentiation protocol by ES/OP9 co-culture system. ....................................................................................... 52
Figure 5. ES cells co-culture with OP9/I-OP9 to induce megakaryocytic differentiation. .............................................................................. 53
Figure 6. Expression of specific genes for megakaryocytes were detected by RT-PCR. .......................................................................................... 54
Figure 7. Expression of cell surface markers on megakaryocytes derived from ES cells co-cultured with OP9/I-OP9 cells. ........................................................................................ 55
Figure 8. Characterization of megakaryocytes derived from ES cells by AchE staining............ 56
Figure 9. Cytotoxictiy of LT on J774A.1 cells. ................................................ 57
Figure 10. LT treatments affect cell size and granularity on TPA-treated HEL cells. ........... 58
Figure 11. Suppressive effects of LT on CD41 expression in TPA-treated HEL cells. ........................................................................................ 59
Figure 12. Polyploid analysis of HEL cells by flow cytometry. ................................. 60
Figure 13. Effects of LT on BrdU incorporation in TPA treated-HEL cells. .......................61
Figure 14. Suppressive effect of LT on CFU-MK formation. ...................................... 62
Figure 15. The experiment outline of in vitro megakaryocytic differentiation using human cord blood-derived CD34+ cells. .................................................................................. 63
Figure 16. Suppressive effect of LT on in vitro megakaryocytic differentiation from cord blood CD34+ cells. ........................................................................................ 64
Figure 17. Quantitative results on the percentage of CD61+ and CD61+/CD42b+ cells in R1 regions. ...................................................................................... 65
Figure 18. LT mediated inhibition the expression of pERK on differentiated cells. ............. 66
Figure 19. LT treatments induced the hypoploid cells during the in vitro differentiation. .............................................................................. 68
Figure 20.Treatments of protective antigen (PA) did not induce apoptosis of human cord blood-derived megakaryocytes. ............................................................................... 69
Figure 21. Platelet isolation and activation test. ............................................ 70
Figure 22. Characterizations of LT-induced hypoploid cells by platelet activation agonists. ..................................................................................... 71
Figure 23. The experimental outline of LT treatments on mouse bone marrow cells. 72
Figure 24. Suppressive effect of LT on mouse bone marrow cells. ............................... 73
Figure 25. TPO treatments reduced LT-mediated mortality in mice. .............................. 74
Figure 26. TPO pretreatments ameliorated LT-mediated thrombocytopenia in mice.................. 75
Figure 27. TPO pretreatments ameliorated LT-mediated megakaryopoiesis suppression in mice. ......................................................................................... 76
Appendices .................................................................................... 77
Reference ..................................................................................... 79

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