臺灣博碩士論文加值系統

大多數人在感染登革病毒時僅有發燒等輕微症狀，但某些病人會產生嚴重的登革出血熱及登革休克症候群。血小板低下症是登革病人常有的症狀，臨床上會有出血的情形。我們過去的研究發現抗登革病毒非結構性蛋白1 (NS1) 的抗體會與人類血小板有交叉反應，進而抑制血小板的凝集作用。根據序列比對，登革病毒NS1蛋白質的C端與宿主的自身抗原序列相似。為了進一步探討NS1上這些具交叉反應之抗原的病理角色，我們將NS1的C端胺基酸271-352切除，產生ΔC NS1抗體，並且比較全長DV NS1抗體與切除C端後的ΔC NS1抗體的病理效應。實驗發現ΔC NS1抗體對血小板的黏附能力較全長DV NS1抗體低，並且不會抑制血小板的凝集作用。而對於抑制血小板凝集功能機制的探討則顯示DV NS1抗體會干擾整合素 (integrin) 的活化。利用小鼠主動免疫模式探討DV NS1抗體造成的出血情形也發現，具全長DV NS1抗體的小鼠較正常小鼠有延長的出血時間，但具ΔC NS1抗體的小鼠則沒有此現象。而被動給予DV NS1抗體會造成小鼠血小板數目下降，且血小板數目下降的時間點與抗體效價消失及抗體黏附在小鼠血小板上的時間相符。然而這些現象在給予ΔC NS1抗體的小鼠身上則不會出現。主動給予DV NS1蛋白或被動給予DV NS1抗體造成小鼠的肝臟病變情形，也不會出現在主動給予ΔC NS1或被動給予ΔC NS1抗體小鼠。總結以上的結果，我們發現了全長DV NS1抗體會造成血小板的功能缺失，小鼠出血時間延長以及肝臟病變，但ΔC NS1抗體則不會。綜合這些發現將可提供登革疫苗研發的新策略。

Dengue virus (DV) infection causes dengue fever, and in some patients may develop into severe dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS). Thrombocytopenia, which is common in dengue fever and is a constant finding in DHF/DSS, is clinically manifested as increased bleeding tendency. Our previous studies showed that antibodies (Abs) against DV nonstructural protein 1 (NS1) cross-reacted with human platelets and inhibited platelet aggregation. Based on the sequence homology alignment, the C-terminal region of DV NS1 protein contained cross-reactive epitopes which are shared between NS1 and self-antigens. To investigate the pathological roles of cross-reactive epitopes of NS1, we compared the effects of Abs against full-length DV NS1 and NS1 lacking the C-terminal amino acids 271 to 352 (ΔC NS1) in this study. We found that anti-ΔC NS1 Abs showed lower platelet binding ability than that of anti-full-length NS1. Anti-full-length NS1 but not anti-ΔC NS1 Abs inhibited ADP-induced platelet aggregation. Studies on the mechanism of platelet aggregation inhibition indicated an effect of anti-DV NS1 Abs on integrin activation. Using a murine model to assess the bleeding tendency caused by anti-DV NS1, we found that the bleeding time in full-length NS1-hyperimmunized mice was longer than that in the normal control. ΔC NS1-hyperimmunized mice showed a bleeding time similar to that of normal control mice. Passive immunization with anti-DV NS1 Abs caused a reduction in platelet number, which was correlated with a decrease in Ab titers and in the binding of Abs to platelets in mice. In contrast, these findings were not observed in mice given anti-ΔC NS1. The liver damage caused by active immunization with DV NS1 protein or passive immunization with anti-DV NS1 Abs were also not detectable in ΔC-NS1 immunized or anti-ΔC NS1-treated mice. In conclusion, we demonstrated that platelet dysfunction, bleeding tendency, and liver abnormality were induced by anti-full-length DV NS1 Abs but not by anti-ΔC NS1 Abs. These findings may provide a strategy for dengue vaccine development.

Chinese Abstract I
English Abstract II
Contents IV
Figure lists VI
Introduction 1
Objective and experimental design 8
1. To investigate the role of cross-reactive epitopes of DV NS1 protein 8
2. To investigate the inhibiting mechanisms of inhibition of ADP-induced platelet aggregation by anti-NS1 9
3. To compare the pathogenic effects of anti-full-length NS1 and anti-ΔC NS1 Abs in vivo 9
A. Material 10
A-1 mice 10
A-2 Cell line 10
A-3 Platelet preparation 10
A-4 Recombinant protein and antibody preparation 10
A-5 Drugs 11
A-6 Antibodies 13
A-7 Consumables 14
A-8 Instruments 15
B. Methods 16
B-1 Cell culture 16
B-2 Platelet binding assay 16
B-3 Platelet aggregation assay 16
V
B-4 Nitric oxide detection 17
B-5 Granule secretion and integrin activation assay 17
B-6 Bleeding time and platelet count 17
B-7 Antibody titer determination 18
B-8 Statistics 18
Results 19
1. Generating the Ab with or without cross-reactive epitopes on DV NS1 protein, and compared the binding ability of these two Abs to endothelial cells and platelets of Abs 19
2. To clarify the mechanism that anti-DV NS1 inhibit ADP-induced platelet aggregation 20
3. Using animal model to explore the pathological role of the cross-reactive epitopes on DV NS1 protein 21
Discussion 23
References 27
Figures 31

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