臺灣博碩士論文加值系統

茲卡病毒屬於黃熱病毒科黃熱病毒屬，目前被發現懷孕中的母親在感染茲卡病毒後，會生出具有神經發育障礙的胎兒，那在正常懷孕過程中保護胎兒腦神經發育中最重要的屏障是胎盤以及血腦障蔽，而目前對於病毒是如何跨越保護胎兒的生理屏障依然是未知的,在本篇研究中我們首先利用胎盤的滋養層細胞株以及人類腦血管內皮細胞株建立體外的屏障系統，首先在胎盤滋養成細胞中我們發現在茲卡感染下的細胞會透過增加緊密連接蛋白的降解，使得細胞和細胞之間的緊密連接被破壞，進而造成病毒顆粒可以透過細胞間隙穿透胎盤細胞障蔽。然而我們同時也發現茲卡病毒可以跨越血腦屏障但卻不會造成細胞之間的緊密連結被破壞，這樣的發現使得我們進一步去研究病毒是否可以透過穿胞運輸來運送病毒顆粒。我們利用螢光標記的茲卡病毒來進行實驗，從我們研究可以發現不論是胎盤細胞屏障或是血管內皮細胞屏障，在加入抑制穿胞運輸的抑制劑後會下降螢光標記病毒跨越障蔽的量，因此本篇研究說明茲卡病毒可通過減少緊密連接蛋白來改變屏障完整性或通過穿胞機制，從而穿過屏障並影響胎兒腦部發育。

Zika virus (ZIKV) infection causes severe neurological symptoms in adults and fetal microcephaly and virus was detected in the brain of microcephaly and meningoencephalitis patient. However, the mechanism of ZIKV crossing the physiological barrier to the central nervous systems (CNS) remains elusive. Placental barrier and blood brain barrier (BBB) protect the fetal from pathogens and ensure healthy brain development during pregnancy. In this study, we used human placenta trophoblasts cells (JEG-3) and human brain-derived endothelial cells (hCMEC/D3) as in vitro models of host barrier. First, ZIKV could infect JEG-3 cells effectively and reduced the amounts of ZO-1 protein and Occludin protein between adjacent cells, suggesting that the permeability of the barrier differentially change in response to ZIKV infection, leading to the virus particle across the host barrier. Interestingly, ZIKV can infect hCMEC/D3 cells without disrupting the BBB barrier permeability and tight junction protein expression. Although no disruption to the BBB was observed during ZIKV infection, ZIKV particles were released on the abluminal side of the BBB model and infected underlying cells. Therefore, we observed that fluorescence-labeled ZIKV particles could cross the in vitro placenta barrier and blood brain barrier model by transcytosis and the action of transcytosis can be blocked by physical and chemical inhibitors. In summary, our study provides new insights that ZIKV can weaken the barrier integrity by reducing tight junction proteins or by transcytosis mechanism in order to cross the barrier and gain access to infect the fetal brain.

誌謝............................i
中文摘要.......................ii
Abstract......................iii
Contents.......................iv
List of Figures...............vii
Introduction....................1
Hypothesis and Specific Aims....8
Materials and Methods...........9
Results........................15
Discussions....................24
References......................27
Figures.........................30

Figure 1 JEG-3 can be infected by ZIKV...30
Figure 2 hCMEC/D3 can be infected by ZIKV...31
Figure 3 ZIKV would affect barrier cells viability...32
Figure 4 ZIKV crosses an in vitro human physiological barrier model and infects vero cells...33
Figure 5 ZIKV damages the placenta barrier in an in vitro transwell model...34
Figure 6 ZIKV infection decreases expression of the tight junction (TJ) protein ZO-1 and occludin in JEG-3 cells by immunofluorescence assay...35
Figure 7 ZIKV infection decreases expression of the tight junction (TJ) protein ZO-1 and occludin in JEG-3 cells by western blot assay...36
Figure 8 ZIKV down-regulated expression of tight junction protein without reduced the mRNA level of these genes...37
Figure 9 ZIKV infection leads to tight Junction protein degradation by proteasomal degradation pathways in placenta barrier...38
Figure 10 Zika virus labeling...39
Figure 11 Transcytosis inhibited at 4°C...40
Figure 12 ZIKV can be transported across the barrier cells monolayer by endocytosis...41
Figure 13 Drugs would not affect JEG-3 cell viability and barrier permeability...42
Figure 14 Effect of individual transcytosis inhibitors on ZIKV transcytosis in JEG-3 cells...43
Figure 15 ZIKV infection does not enhanced permeability of the endothelial cell monolayer...44
Figure 16 ZIKV infection did not disrupt tight junction complex in hCMEC/D3 cells...45
Figure 17 Drugs would not affect hCMEC/D3 cell viability and barrier permeability...46
Figure 18 Effect of individual transcytosis inhibitors on ZIKV transcytosis in hCMEC/D3 cells...47
Figure 19 ZIKV transport can be blocked by endocytosis inhibitors...48

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