||The infectivity and effects of dengue viruses on human primary lung epithelium and carcinoma cells
||Institute of Molecular Medicine
lung epithelial carcinoma
肺的積水、水腫、出血和急性呼吸道窘迫症發生在一些登革病毒感染的病人中。在登革病人的肺組織中能夠測到登革病毒的核酸和病毒的抗原，顯示登革病毒可能也會感染人類的肺部組織。本研究中以四種不同血清型登革病毒感染代表六種不同部位的肺癌細胞，以及正常肺部組織細胞。利用免疫螢光及免疫組織化學染色，首先證實登革病毒能在六種不同的肺癌細胞和人類正常肺上皮組織中被偵測到。藉由穿透式電子顯微鏡，可以在粗糙內質網中進一步看到登革病毒的顆粒。此外，以DIG-狹縫雜交(DIG-slot blotting)和即時反轉錄聚合連鎖反應(real time RT-PCR)的偵測，進一步證實登革病毒在肺細胞中具有複製和產出病毒的能力。綜合以上所述，肺上皮細胞極可能是一個登革病毒感染時的目標之一。為了說明登革病毒對感染宿主細胞的細胞激素(cytokines)和化學激素(chemokines)的影嚮。進一步以反轉錄聚合連鎖反應(RT-PCR)觀察登革病毒感染之肺細胞中IFN-�, IL-1�, IL-6, IL-8, TGF-�, TNF-�, RANTES和VEGF...等細胞激素(cytokines)和化學激素(chemokines)的mRNA表現。結果顯示，只有在野生型病毒的感染下，IL-6, IL-8 and RANTES的mRNA表現明顯上升；酵素連結免疫吸附分析(ELISA)的方式，在蛋白質的階層也看到了相同的結果。這些結果和臨床登革出血熱／登革休克症狀具有IL-6、IL-8和RANTES高度表現是相同的。最後我們也證實登革病毒誘導肺細胞IL-6的產生，主要經由NF-κB的路徑來進行的。結論是，我們認為在登革病人之肺部，可能產生IL-6、IL-8和RANTES，並在登革病毒感染所引發之肺水腫扮演重要的角色。
Pulmonary effusion/edema, hemorrhage and acute respiratory distress syndrome may occur in some of the patients with dengue viruses infection. The detection of dengue viruses and RNA in the patients’ lung tissue suggests that dengue virus could infect human lung cells. This study was designed to examine the infectivity of dengue viruses in human lung tissue and their effects on the host cells. A total of six lung epithelial cancer cell lines and primary human lung tissue were used to test with four serotypes of dengue viruses. We demonstrated that dengue viruses can permissively infect both primary human lung epithelial cells and six lung cancer cell lines by immunofluorescent and immunohistochemical staining. The viral particles were clearly demonstrated in the rough endoplasmic reticulum (RER) of infected cells under transmission electron microscopy. The viral RNA synthesis and viral production were analyzed by DIG-slot blotting and real time RT-PCR in these cells, respectively. Altogether, our data demonstrate that lung epithelial cells are possible targets of dengue viruses in vivo. Further study on the effect of virus infection on lung epithelial cells revealed the RNA expression of IL-1�, IL-6, IL-8, TGF-�, TNF-�, VEGF, RANTES and IFN-� by RT-PCR. There was remarkable increase of IL-6, IL-8 and RANTES expression by RT-PCR and ELISA in virus infected cells. These results are consistent with clinical observation that IL-6, IL-8 and RANTES were highly expressed in DHF/DSS patients. In addition, the IL-6 induced by DEN-2 virus infection is dependent on NF-κB. In conclusion, our data suggest that IL-6, IL-8 and RANTES play an important role in the pulmonary effusion of patients with dengue virus infection.
1. Index of Tables I
2. Index of Figures II
3. Abbreviation IV
4. Introduction 1
5. Materials and Methods 8
6. Results 16
7. Discussion 20
8. References 24
9. Appendix of Tables 31
10. Appendix of Figures 35
11. Curriculum Vitae 63
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