臺灣博碩士論文加值系統

登革病毒 (Dengue virus; DENV) 是一種藉由蚊子傳播的黃熱屬病毒，且全球平均每年約有3.9億的人口會被登革病毒感染。DENV除了能夠造成大家所熟知的典型登革熱 (dengue fever) 外，也可能引起嚴重甚至會致死的登革出血熱 (dengue hemorrhagic fever, DHF) 或登革休克症候群 (dengue shock syndrome, DSS)。患有DHF或DSS的病人常會出現一些典型的病徵包括異常出血及血小板數量低下。登革病毒非結構性蛋白1 (nonstructural protein 1, NS1) 是一個在登革感染過程中會出現在患者血液中的蛋白且在先前有不少篇研究證實DENV NS1能夠造成血管滲漏。然而，是否DENV NS1也會造成血小板數量低下則仍待釐清。先前的研究發現在登革病人血液中的血小板有增加活化與凋亡的現象，並且與病人血小板數量的減少呈現正相關。因此我們認為很有可能血液中的DENV NS1蛋白會引起血小板的活化與凋亡並進而造成血小板數量低下。為了驗證這個假說，我們使用由果蠅所產的重組DENV NS1蛋白來與正常人的血小板進行反應，並檢查是否會造成血小板的活化與凋亡。首先，我們發現DENV NS1可以黏附到血小板的表面藉由與血小板表面的類鐸受體 4反應。接著，我們發現DENV NS1能夠使血小板的活化指標P-selectin大量表現，而這樣的現象則可以被針對NS1的單株抗體所抑制。此外，血小板在加入低劑量的血小板刺激劑ADP前如果有接受DENV NS1的刺激，血小板的凝集能力也會有增強的現象。同時，我們也發現DENV NS1能夠造成血小板的凋亡包括引發一些細胞凋亡的現象：phosphatidylserine的外翻、caspase 9跟3的活化及血小板粒線體中的活性氧化物質 (Reactive oxygen species, ROS) 製造增加；而且DENV NS1可以刺激血小板分泌巨噬細胞移動抑制因子(macrophage migration inhibitory factor, MIF)，MIF已知與血管通透性增加有關。最後我們更發現DENV NS1能夠增加血小板對白血球的黏附，尤其和嗜中性球。總括以上的結果，我們發現DENV NS1能夠引發血小板活化與凋亡，並且可能對於登革病毒感染所造成的血小板數量低下扮演一定的重要角色。

Dengue virus (DENV), a mosquito-borne flavivirus, is responsible for ~390 million infections annually, and leads to dengue fever or potentially lethal dengue hemorrhagic fever (DHF) or shock syndrome (DSS). DHF/DSS patients often show symptoms such as vascular leakage and thrombocytopenia. During dengue infection, DENV nonstructural protein 1 (NS1) can be secreted into blood and causes vascular leakage. However, whether DENV NS1 is involved in the pathogenesis of thrombocytopenia remains unclear. In previous study, it has been shown that low platelet counts coexist with high platelet activation and apoptosis in dengue patients. In this study, we hypothesized that DENV NS1 may play a critical role to induce platelet activation and apoptosis, which may contribute to thrombocytopenia during dengue infection. To test this hypothesis, freshly-isolated platelets from healthy donors were treated with DENV recombinant NS1 from drosophila and were evaluated for markers of activation and apoptosis. First, our result showed that DENV NS1 can bind to platelets via interacting with Toll-like receptor 4 (TLR4) on platelet surface. Then, we found that the platelet activation marker, P-selectin, was highly expressed in DENV NS1-stimulated platelets and the increased expression could be inhibited by anti-NS1 monoclonal antibody. Furthermore, DENV NS1 pretreatment enhanced platelets aggregation induced by suboptimal dose of ADP stimulation. Moreover, DENV NS1-stimulated platelets exhibited signs of apoptosis such as increased surface phosphatidylserine exposure, mitochondrial ROS production and activation of caspase-9 and -3. Interestingly, the secretion of macrophage migration inhibitory factor (MIF), a cytokine which may increases vascular permeability, was also increased in DENV NS1-stimulated platelets. Finally, we found that DENV NS1 can also increase adhesion of platelets to leukocytes, particular neutrophils. Taken together, our results suggest that DENV NS1 could directly induce platelet activation and apoptosis, which may contribute to the pathogenesis of thrombocytopenia during dengue infection.

中文摘要 I
Abstract III
Acknowledgement V
Table of Contents VI
List of Figures IX
Abbreviations Index X
1. Introduction 1
1.1. Dengue virus 1
1.1.1. Dengue virus infection: Epidemiology 1
1.1.2. Characteristics of dengue virus 2
1.1.3. DENV nonstructural protein 1 3
1.1.4. Clinical symptoms and diagnostic methods of DENV infection 5
1.2. Thrombocytopenia in dengue 6
1.2.1. Characteristics of platelet 7
1.2.2. The possible mechanisms of thrombocytopenia in dengue 9
2. Objective and Specific Aims 12
1. To determine whether DENV NS1 can induce platelet activation. 12
2. To determine whether DENV NS1 can induce platelet apoptosis. 13
3. To determine whether DENV NS1 can increase adhesion of platelets to leukocyte 13
4. To determine whether DENV NS1-stimulated platelet may increase endothelium permeability 13
3. Materials and Methods 14
3.1. Materials 14
3.1.1. Proteins 14
3.1.2. Reagents 14
3.1.3. Antibodies 16
3.1.4. Consumables 17
3.1.5. Instruments 18
3.2. Methods 19
3.2.1. Isolation of peripheral blood platelets and leukocytes 19
3.2.2. In vitro platelet stimulation 20
3.2.3. Flow cytometric analysis for DENV NS1 binding 20
3.2.4. Flow cytometric analysis for P-selectin expression 21
3.2.5. Flow cytometric analysis for phosphatidylserine exposure 21
3.2.6. Flow cytometric analysis for mitochondrial-derived reactive oxygen species (ROS) production 21
3.2.7. Platelet aggregation test 22
3.2.8. Indirect immunofluorescence assay 22
3.2.9. SDS-PAGE 22
3.2.10. Western blot 23
3.2.11. ELISA 23
3.2.12. Liu’s stain 23
3.2.13. Statistical analysis 24
4. Results 25
4.1. DENV NS1 binds to platelet. 25
4.2. Colocalization of DENV NS1 and TLR4 on platelets surface. 25
4.3. DENV NS1 induces platelet activation. 26
4.4. Signal pathway other than TLR4 may also involve in DENV NS1-induced platelet activation. 27
4.5. DENV NS1 induces apoptosis of platelet. 28
4.6. NS1-increased P-selectin expression can be inhibited by ROS inhibitor. 29
4.7. DENV NS1 increases platelet adhesion on leukocyte. 30
4.8. DENV NS1-stimulated platelet may increase endothelium permeability. 31
5. Discussion 33
6. Conclusion 40
7. References 41
8. Figures 54

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