臺灣博碩士論文加值系統

根據世界衛生組織估計，登革病毒 (Dengue virus) 在全球每年約造成五千萬至一億個病例發生。登革病毒感染的症狀可以區分為較輕微的登革熱，較嚴重的登革出血熱和登革休克症狀，而血小板低下普遍存在於這些症狀當中。巨核細胞為血小板的前驅細胞，巨核細胞的生成包含了巨核細胞分化及成熟的過程，最終會分裂成血小板釋放至周邊血液。過去的研究顯示，登革病毒會抑制巨核細胞的生成，但登革病毒是如何造成巨核細胞的生成受到抑制，並且是透過哪個分子產生影響的，病毒的複製是否是必須的，目前還尚未釐清。登革病毒套膜蛋白(dengue viral envelope protein domain III) 是登革病毒負責跟宿主細胞結合的片段，進而幫助病毒進入到宿主細胞內。在本篇研究當中發現登革病毒套膜蛋白可以抑制巨核細胞的生成，包括抑制TPA (12-O-tetradecanoylphorbol-13-acetate) 誘導的HEL (Human erythroleukemia) 細胞株、細胞激素刺激由臍帶血純化的血球幹細胞及血小板生成素 (TPO, thrombopoietin) 誘導的老鼠骨髓細胞往巨核細胞分化。同時發現，經由眼窩靜脈叢注射登革病毒套膜蛋白至小鼠體內，可以達到像注射登革病毒一樣，誘發相似程度的巨核細胞數量的減少。並且更進一步證明登革病毒套膜蛋白可以直接結合至巨核細胞，及其引起的巨核細胞生成抑制，與病毒複製無關，而與巨核細胞的自噬作用(autophagy)失調及細胞凋亡有關係。本篇研究得到的結論是登革病毒套膜蛋白與巨核細胞結合引發的自噬作用失調及細胞凋亡，參與了登革病毒所引起的血小板低下。因此，登革病毒套膜蛋白或許可以做為藥物開發的標的來減緩登革病毒感染所造成的血小板低下症狀。

Dengue virus (DENV) causes 50-100 million infections annually worldwide. Thrombocytopenia is the common feature of dengue viruses-induced mild dengue fever, more severe dengue hemorrhage fever, and dengue shock syndrome. Megakaryopoiesis is the differentiation and maturation processes of megakaryocytes which are the precursors of platelets. Previous studies have demonstrated that DENV can induce thrombocytopenia by megakaryopoiesis suppression; however, the mechanism for megakaryopoiesis suppression, such as whether viral replication is required or what molecule of DENV involved is still unclear. DENV envelope protein domain III (DENV-EIII) is responsible for binding to the host cells. This study found DENV-EIII could suppress TPA (12-O-tetradecanoylphorbol-13-acetate)-induced megakaryocytic differentiation of human erythroleukemia cells, cytokines-triggered megakaryocytic differentiation of CD34+ cells derived from human umbilical cord blood, and thrombopoietin-induced megakaryocytic differentiation of mice bone marrow cells. Megakaryopoiesis suppression was also found in the bone marrow of DENV-EIII-injected C57BL/6J mice like DENV-injected mice. Further, this study demonstrated that DENV-EIII-induced megakaryopoiesis suppression is caused by
DENV-EIII directly binding to megakaryocytes and triggering autophagy impairment and apoptosis, and is independent of viral replication. In conclusion, DENV-caused thrombocytopenia is mediated by DENV-EIII-elicited megakaryopoiesis suppression through binding to megakaryocytes and inducing autophagy impairment and apoptosis. Therefore, DENV-EIII could be developed as a drug target to ameliorate thrombocytopenia in dengue patients.

致謝................................................................................................................................. I
中文摘要....................................................................................................................... III
Abstract .........................................................................................................................IV
Content ...........................................................................................................................V
Introduction .................................................................................................................... 1
1. Dengue ................................................................................................................ 1
1.1. Dengue epidemic ............................................................................................. 1
1.2. Structure of dengue virus ................................................................................. 1
1.3. Receptors for dengue virus entry into host cells .............................................. 3
1.4. Clinical symptoms of dengue infection ........................................................... 3
2. Megakaryopoiesis ...................................................................................................... 4
2.1. Commitment of progenitor to megakaryocytes ............................................... 4
2.2. Maturation of megakaryocytes ........................................................................ 5
2.3. Regulation of megakaryopoiesis ...................................................................... 5
Aims of this project ........................................................................................................ 7
Materials and Methods ................................................................................................... 9
Results .......................................................................................................................... 25
1. Purification of DENV-EIII protein ................................................................ 25
2. DENV-EIII suppressed megakaryocytic differentiation of HEL cell line ..... 25
3. DENV-EIII suppressed megakaryopoiesis of progenitor cells from cord
blood ..................................................................................................................... 27
4. DENV-EIII suppressed megakaryopoiesis from mouse bone marrow cells .. 29
5. DENV-EIII suppressed megakaryopoiesis in vivo ......................................... 30
6. DENV-EIII suppressed megakaryopoiesis by directly binding to MKs ........ 31
7. DENV-EIII suppressed megakaryopoiesis in CD34+ cells from human cord
blood by apoptosis and autophagy impairment .................................................... 31
Discussion .................................................................................................................... 33
Perspective ................................................................................................................... 38
Figures.......................................................................................................................... 40
Figure 1. Purification of DENV-EIII protein with extremely low endotoxin
contamination........................................................................................................ 41
Figure 2. Purification of GST protein with low endotoxin contamination. .......... 42
Figure 3. No obvious effect of DENV-EIII on TPA-treated HEL
cells-megakaryocytic marker expression. ............................................................. 43
Figure 4. Suppressive effect of DENV-EIII on TPA-treated HEL
cells-polyploidization............................................................................................ 45
Figure 5. Suppressive effect of DENV-EIII on colony forming
unit-megakaryocytic assay from human cord blood derived mononuclear cells.. 47
Figure 6. Cytokines-induced megakaryopoiesis of CD34+ cells derived from
human cord blood. ................................................................................................ 50
Figure 7. Suppressive effect of DENV-EIII on cytokines-induced
megakaryopoiesis of CD34+ cells derived from human umbilical cord blood. .... 54
Figure 8. Suppressive effect of DENV-EIII on megakaryopoiesis from mouse
bone marrow cells. ................................................................................................ 56
Figure 9. Suppressive effect of DENV on megakaryopoiesis in vivo. .................. 58
Figure 10. Suppressive effect of DENV-EIII on megakaryopoiesis in vivo. ........ 60
Figure 11. Binding of DENV-EIII on megakaryocytic surface. ........................... 62
Figure 12. DENV-EIII-induced megakaryopoiesis suppression by impaired
autophagy and apoptosis. ...................................................................................... 64
Supplementary Table 1. Clinical manifestations of Dengue hemorrhagic fever... 65
Supplementary Figure 1. Dengue viral genome and structure of dengue envelope
protein. .................................................................................................................. 66
Supplementary Figure 2. TPO-induce megakaryocyte development. .................. 67
Supplementary Figure 3. Competitive blocking of a dose range of DENV-EIII on
DENV infection and replication in BHK21 cells. ................................................ 68
Supplementary Figure 4. Infection and replication of DENV in BHK21 cells, but
not in mMK. .......................................................................................................... 70
Supplementary Figure 5. Cytotoxicity effect of anti-MK fraction purified from
anti-NS1 on differentiated megakaryocytes-derived from mice bone mononuclear
cells. ...................................................................................................................... 72
References .................................................................................................................... 73

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