臺灣博碩士論文加值系統

登革病毒是RNA病毒的一種，它被歸類到黃病毒科，黃病毒屬中，共有四種血清型。人感染登革病毒可能會發展成如登革出血熱或登革休克症等的致命性併發症。本論文利用兩種免疫細胞：T細胞與單核球細胞研究登革二型病毒感染以後不同免疫細胞所表現出的各種差異。分析的重點為細胞的成長、死亡。我們使用一組登革二型病毒特異性的核酸序列為引子於聚合酵素連鎖反應中證實病毒可以在所有研究的細胞中複製，並且用隙縫點墨法來定量病毒RNA的在細胞內的量，以及免疫螢光染色觀察病毒蛋白的表現。我們發現，登革病毒感染吸附單核球及T細胞株的能力類似，但是在單核球內的複製能力較T細胞差。受登革二型病毒感染後，Molt-4和Jurkat兩株T細胞株的生長受壓制；反之在單核球細胞株中，U-937細胞株的生長速率加快，而HL-60細胞株的生長則不受病毒感染所影響。另一方面，以death-ELISA and Merocyanine 540 binding assay來分析細胞的計畫性死亡，結果顯示Molt-4和Jurkat兩株T細胞株在受登革二型病毒感染後48小時，細胞計畫性死亡速度增加，然而在U-937 和HL-60兩株單細胞株卻不受影響。在死亡相關基因表現的研究上，發現Bcl-2的 mRNA量並不因病毒感染而有太大改變。但是T細胞內Fas-L及Fas的mRNA則受病毒感染而有增加。綜合以上結果，雖然這些細胞皆可受登革二型病毒感染，但是不同類的細胞反應則各有不同。

Infection with dengue virus, an RNA virus classified to flaviviridae, may develop life-threatening complications such as dengue hemorrhagic fever and dengue shock syndrome. A variety of human mononuclear cell lines can be infected with dengue virus in vitro. In this study we compared the responses of T cells and monocytes after dengue-2 virus infection to determine their possible roles in the pathogenesis. Cell growth and death were analyzed. Reverse transcription-polymerase chain reaction analysis using primers specific to dengue viral RNA confirmed the replication of dengue-2 virus in cells. Viral RNA and viral E protein was analyzed by Slot blotting and immunostaining, respectively. We found that lesser viral RNA and viral protein were detected in monocytes than that in T cells. Futhermore, dengue-2 virus infection suppressed the growth of Molt-4 and Jurkat cells, while enhanced it of U-937 cells. The growth of HL-60 cells was not affected by dengue-2 virus infection. An increase in apoptosis, determined by death-ELISA and Merocyanine 540 binding assay, in Molt-4 and Jurkat cells was observed after 48-hr virus infection. Dengue virus did not enhance apoptosis in HL-60 and U-937 cells. Moreover, we found that there was more fas-L and fas mRNA, but not bcl-2, in virus infected cells. In conclusion, different cells might have distinct responses to dengue-2 virus infection.

中文摘要 I
英文摘要 III
誌謝 IV
目錄 V
圖目錄 VII
符號及縮寫 VIII
主文
I、前言 1
II、材料 6
A、 細胞株 6
B、登革二型病毒 6
C、培養基及相關藥品 6
D、試劑 7
E、器材 8
III、方法 11
A、T細胞株 (Molt-4＆Jurkat cells)與單細胞株
(HL-60＆U-937 cells)培養的狀況 11
B、細胞感染登革熱二型病毒後的成長情況 13
C、反轉錄酵素-聚合酵素連鎖反應 15
D、免疫螢光染色法 19
E、隙縫點墨法 21
F、細胞計畫性死亡的測定 24
IV、結果 30
一、 登革二型病毒在細胞中的感染與複製情形 30
二、 登革二型病毒蛋白在細胞中的表現 32
三、 登革二型病毒對細胞生長所造成的影響 33
四、 登革二型病毒的感染可造成T細胞株進行細胞凋亡 34
五、 登革二型病毒造成T細胞株進行細胞凋亡中的相關性
基因表現的偵測 35
V、討論 36
VI、參考文獻 42
圖 49
實驗流程 69
作者經歷 70
圖目錄
Fig. 1 DEN-2 virus RNA deteted by RT-PCR. 49
Fig. 2 DEN-2 virus RNA deteted by nest PCR. 50
Fig. 3 Slot blotting analysis of DEN-2 viral negative
stranded RNA. 51
Fig. 4 Immunofluorecent staining of DEN-2 viral E protein.
52
Fig. 5 Immunofluorecent staining of DEN-2 viral E protein.
53
Fig. 6 Percentage of DEN-2 viral E protein positive cells in
immunofluorecent staining. 54
Fig. 7a Representative histogram of Jurkat cells at 96 hr-
postinfection. 55
Fig. 7b Immunofluorecent staining of DEN-2 viral E protein.
56
Fig. 8a Representative histogram of U-937 cells at 96 hr-
postinfection. 57
Fig. 8b Immunofluorecent staining of DEN-2 viral E protein.
58
Fig. 9 Effect of DEN-2 virus infection on T cell lines growth. 59
Fig. 10 Effect of DEN-2 virus infection on monocytic cell
lines growth. 60
Fig. 11 A representative histogram of MC540 stainingof Jurkat
cell after DEN-2 virus infection. 61
Fig. 12 Preapoptosis detection in Molt-4 cells. 62
Fig. 13 Preapoptosis detection in Jurkat cells. 63
Fig. 14 Preapoptosis detection in HL-60 cells. 64
Fig. 15 Preapoptosis detection in U-937 cells. 65
Fig. 16 Apoptosis detection by death-ELISA assay. 66
Fig. 17 RT-PCR for determining mRNA expression of death-
related genes. 67
Fig. 18 RT-PCR for determining mRNA expression of death-
related genes. 68

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