臺灣博碩士論文加值系統

本篇論文中，利用動物模式探討A型流感病毒中 PB1-F2蛋白質，在病毒感染宿主所引發的免疫反應中所扮演的角色。PB1-F2 蛋白質是由A型流感病毒聚合酶蛋白PB1 (Polymerase protein, Basic) mRNA經由一額外的開放讀碼區 (alternative open reading frame) 轉譯出來，所以稱為PB1-F2。目前已知PB1-F2會藉由改變粒線體內膜的通透性，增強流感病毒感染時所造成的細胞凋亡 (Apoptosis) 效應，但這個現象僅在基因重組PR8病毒株感染的U937單核球 (Human leukemic monocyte lymphoma cell line) 觀察到。我們利用逆向遺傳法 (reverse genetics) 產生不表現PB1-F2蛋白質的基因重組病毒，這個點突變位在WSN病毒第二段RNA基因組的第120個核酸，由於PB1與PB1-F2利用不同的開放讀碼區轉譯，所以點突變的結果將會造成PB1-F2起始密碼由MetThr、不再表現PB1-F2，可是對於PB1則是一個不影響PB1胺基酸序列及表現的啞突變 (silent mutation) 。在動物實驗的部份我們利用 ICR 或 BALB/c 分別感染基因重組野生型病毒 (rWSNwt) 或 PB1-F2缺失型病毒 (rWSNPB1-F2-) ，並在感染後特定的時間點觀察小鼠死亡率、體重變化；另一方面收取病毒感染後小鼠的脾臟細胞，以免疫細胞表面抗原抗體進行免疫染色後，再利用細胞流式儀測量各種免疫細胞此兩株病毒感染後一到六日間、每隔兩日免疫細胞群體分布的變化。
在我們的實驗中，以rWSNwt病毒在感染U937 細胞並不會觀察到之前研究者以PR8基因重組病毒感染時、增強病毒引起的細胞凋亡；而在動物實驗中，我們發現rWSNPB1-F2- 病毒感染小鼠會引起較嚴重的病徵 (例如：體重減輕較多) 及較高的死亡率 (較低的MLD50 病毒力價) 。除此之外rWSNPB1-F2- 病毒會增加帶有以下數種細胞表面抗原的免疫細胞在脾臟細胞中所佔的比例，如：CD11b+ (巨噬細胞；macrophages) 、CD11c+ (樹突狀細胞； dendritic cells) 、B220+ (B 細胞) 、CD80+ (抗原表達細胞； Antigen presenting cells) 及自然殺手細胞 (NK cells) 。綜觀以上結果，PB1-F2 對於rWSNwt病毒株來說，並不會增強病毒感染所造成的U937 細胞凋亡，但在動物模式下，缺少PB1-F2的基因重組WSN病毒株會提升數種免疫細胞的表現，可能進而增強病毒感染所引發的過度免疫反應，因而導致宿主病徵加重、致死率提高。

In this study, we applied animal model to understand the role of viral protein PB1-F2 in influenza A virus infection. The PB1-F2 protein is translated by an alternative open reading frame of Influenza virus A polymerase gene, such as PB1. It has been shown that PB1-F2 can induce cellular apoptosis in certain cell type, monocyte. By reverse genetic, we have generated a mutant recombinant influenza A virus WSN strain lacking PB1-F2. A silent mutation was introduced by a T120C nucleotide substitution in PB1 gene to eliminate the translation of PB1-F2. Four weeks old ICR or BALB/c mice were infected with recombinant wild type virus (rWSNwt) or PB1-F2 deficient mutant viurs (rWSNPB1-F2-). The virus lethal doses, mice weight loss were then measured at different time points of post infection. The splenocytes of infected animals were also collected and subjected to immunostaining and flowcytometry for examination of the lymphocyte population.
According to literatures, in PR8 strain virus, PB1-F2 can induce specific apoptosis of U937 cells (a human leukemic monocyte lymphoma cell line). However, on our hands, rWSNwtand rWSNPB1-F2- showed no difference in inducing apoptosis in infected-U937 cells. Nevertheless, via animal model, we found rWSNPB1-F2- virus can cause severer illness (more weight loss) and lower MLD50 than rWSNwt , which indicates rWSN virus lacking PB1-F2 caused higher pathogenicity. Moreover, infection of rWSNPB1-F2- virus increased CD11b+ (macrophages) , CD11c+ (dendritic cells) , B220+ (B cells) , CD80+ (Antigen presenting cells) and NK cells populations more than that of rWSNwt virus in four weeks old BALB/c mice at day 4 post-infection. Taken together, we conclude that the expression of PB1-F2 in influenza A virus WSN strain did not have obvious effect on apoptosis in U937 cells. rWSNPB1-F2- virus infection can induce several kinds of the host immune cells more than rWSNWt infection in mice.

第一章 A型流感病毒 - 1 -
第一節 A型流感病毒與宿主免疫反應 - 2 -
第二節 A/WSN/33病毒株與宿主免疫反應 - 3 -
第三節 A型流感病毒與細胞凋亡 - 4 -
第四節 A型流感病毒與逆向遺傳法基因重組病毒 - 6 -
第二章 研究材料與方法 - 8 -
第一節 細胞與病毒 - 8 -
a. 細胞： - 8 -
b. 高力價病毒的製備： - 8 -
c. 病毒在MDCK細胞中複製的速率： - 9 -
d. rWSNWt病毒及rWSNPB1-F2-突變株病毒對U937細胞凋亡的影響： - 9 -
第二節 動物實驗模型觀察rWSNWt病毒及rWSNPB1-F2-突變株病毒感染個體所造成的差異 (17, 25) ： - 10 -
a. 細胞與病毒： - 10 -
b. MLD50測量： - 10 -
c. 病毒對小鼠體重變化的影響： - 11 -
d. 病毒在宿主體內複製情形的觀察： - 11 -
e. 脾臟細胞的免疫染色： - 11 -
f. 血球凝集試驗 (HA test) 及血球凝集抑制試驗 (HI test) ： - 12 -
第三章 實驗結果 - 14 -
第一節 基因重組病毒特性體外測試 - 14 -
a. PB1-F2不參與rWSN病毒株的生長及繁殖 - 14 -
b. rWSNWt與rWSNPB1-F2-感染引發U937單核球細胞凋亡的程度相近 - 14 -
第二節 基因重組病毒動物實驗模型 - 15 -
a. rWSNWt與rWSNPB1-F2-感染在小鼠體內的生長及繁殖的情況相似 - 15 -
b. 比較rWSNWt與rWSNPB1-F2-對小鼠的致病性及致死率 - 15 -
c. PB1-F2會抑制受rWSNWt感染小鼠脾臟中多種免疫細胞的活化 - 16 -
第四章 討論 - 35 -
第三節 單核球細胞凋亡的實驗結果 - 35 -
a. 基因重組病毒取得的方式不同 - 35 -
b. 病毒培養的方式不同 - 36 -
c. 基因重組病毒的來源病毒株不同 - 36 -
第四節 病毒感染造成單核球細胞凋亡的原因 - 37 -
第五節 動物實驗的結果 - 38 -
第六節 流感病毒感染與免疫反應 - 38 -
a. 流感病毒感染的起始反應 - 39 -
b. 流感病毒感染對樹突狀細胞的影響 - 39 -
c. 流感病毒感染對巨噬細胞的影響 - 40 -
d. 延伸實驗 - 40 -
參考文獻 - 43 -

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