|
第一部份：
登革熱第二型病毒感染肝細胞及內皮細胞引發RANTES活化的訊息傳遞研究
登革熱病毒屬於黃熱病毒屬(Flaviviridase)的一員，全球每年感染登革病毒的人口超過12億人，並且有一部份人會引發更嚴重的登革出血熱(Dengue hemorrhagic fever, DHF)及休克型登革熱(Dengue shock syndrom, DSS)。
在in vitro及in vivo的研究中已證實：感染登革熱病毒會引發包含RANTES的各種細胞趨化素(chemokines)，並藉以引起肝臟的發炎反應；然而,造成這些細胞趨化素活化的訊息傳遞仍然不清楚。在我們的研究中發現：當登革二型病毒感染肝臟細胞及內皮細胞時，利用西方墨點法(Western blot)我們發現細胞內的mitogen-activated protein kinase (MAPKs) p38, extracellular signal-regulated kinase (Erk), 及c-jun-NH2-terminal kinase (JNK)會被磷酸化(Phosphorylation)而活化，利用Dominant negative mutant的ras, raf-1, p38, Erk和JNK的質體(Plasmid)，更進一步證實我們的先前結果，根據以上的發現，感染登革二型病毒會經由Ras, Raf-1, JNK, Erk及p38等相關的訊息傳遞路徑來活化RANTES。為了更進一步探討登革病毒的單一基因(prM, core, E及NS1)對RANTES活化的影響，我們利用可誘導的質體(Indcuible plasmid)來建構攜帶登革二型病毒單一基因(prM, core, E及NS1)的質體，然而我們發現登革二型病毒的這些單一基因並不會引起RANTES的活化，未來我們將針對引發RANTES活化的訊息傳遞路徑，利用專一性的抑制劑(Inhibitor)來觀察RANTES的活化對招募免疫細胞所扮演的角色。這些知識的累積將有助於利用減少RANTES的產生來降低發炎現象以期發展為一有效的治療方法。
第二部份：
建立新生鼠系統以利用登革二型病毒的全長cDNA迅速地篩選及大量地生產具傳染力的登革二型病毒
這是第一個使用新生鼠腦並利用登革二型病毒的全長cDNA迅速地篩選及大量地生產具傳染力的登革二型病毒的研究報告，我們從Dr. B. Falgouth得到一個具有感染性登革二型病毒的全長cDNA質體(pRS424FLD2NCG)並將此質體送入大腸桿菌(STBL-2 strain)中，以限制脢(SacI, EcoRI及KpnI)切割，確定此質體確實帶有登革二型病毒全長cDNA的基因；以此cDNA為模板利用in vitro transcription合成登革二型病毒的全長RNA，並將此RNA以腦內注射方式注入六天大的新生鼠(ICR strain)腦中來證實此RNA確實具有感染性，經腦內注射具感染性登革二型病毒RNA的新生鼠會在注射後第13天發生後肢癱瘓現象，然後取出此新生鼠的腦部組織，利用免疫染色法(Immunochistochemistry)及抗登革二型病毒core, E及NS1(Anti-core, anti-E及anti-NS1 monoclone antibody)抗體加以證實鼠腦內具有感染性登革二型病毒存在；為進一步證實確實具有感染性登革二型病毒存在，我們將鼠腦萃取液與蚊子細胞C6/36混和培養，三天後發現產生CPE (Cytopathic effect)現象，將C6/36的細胞萃取物以登革病毒非結構性蛋白質(NS1)專一性primers (NS1F01/NS1R01)利用RT-PCR方法證實登革病毒的存在。並利用限制脢(BstEII)切割，以證實鼠腦內所分離出的登革病毒乃由有感染性登革二型病毒的全長cDNA質體所產生出來的。此外以此種方式從鼠腦中所得到的病毒數約有2.02×109 (PFU/g)，勝於之前以細胞株(C6/36，BHK及LLC-MK2)送入具感染性登革二型病毒RNA所產生的病毒數為多。因此我們成功地建立一套新生鼠系統以用來迅速地篩選及大量地生產具傳染力的登革二型病毒，另外，此套系統將可用於登革病毒基因分子層次的研究。
|
|
Part Ι. Signal transduction of RANTES activation in dengue-2 virus infected hepatocyte and endothelial cells.
Dengue virus belonging to the Flaviviridase infects more then twenty billion people worldwide each year and causes sever dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Dengue viruses involve in liver inflammatory responses mediated by expressing various chemokines including RANTES has been confirmed in vitro as well as in vivo. However, the underline signaling remains unknown. Here, we found that mitogen-activated protein kinase (MAPKs) p38, extracellular signal-regulated kinase (Erk), and c-jun-NH2-terminal kinase (JNK) can be phosphorylated and activated in dengue-2 virus infected hepatocyte and epithelial cells demonstrated by western blotting. Dominant negative mutants of ras, raf-1, p38, Erk, and JNK further confirm our previous results. Taken-together, dengue-2 virus utilizes Ras, Raf-1, JNK, Erk and p38 related signal pathways to active RANTES. Further analysis the involvement of the individual genes prM, core, E and NS1 in RANTES activation, the inducible plasmids harboring the viral genes were constructed. However, no effect was detected among the genes tested. The observation of the role of RANTES in recruiting immune cells by using a transwell assay is underway. The knowledge gathered will shed light on a useful therapeutic approach in blocking inflammatory infiltrates by decreasing RANTES expression.
Part ΙΙ. Establishment of a suckling mice system to rapidly screen and massively produce infectious dengue-2 viruses from full-length cDNA.
This is the first report to use suckling mice brain to screen and massively produce infectious dengue virus from full-length cDNA plasmid. An infectious full-length cDNA plasmid (pRS424FLD2NCG) of dengue-2 virus (New Guinea C strain) obtained from Dr. B. Falgout was used to transform E. coli STBL-2 strain. The plasmid harboring full-length dengue-2 virus cDNA was confirmed by restriction endonucleous digestion (SacI, EcoRI and KpnI). Dengue-2 virus full-length RNA was synthesized from the cDNA template by in vitro transcription. The infectivity of the RNA was then tested by inoculating intracerebrally (IC) the RNA into 6 days suckling mice (ICR). The mice with infectious RNA showed apparent paralysis symptom around day 13 post-injection. The infected mice were sacrificed and the brain was sectioned. The existence of infectious dengue virus was confirmed by immunohistochemistry staining using anti-E, anti-core and anti-NS1 monoclonal antibodies. To further verify the production of mature dengue-2 virus, the brain extract was collected and co-cultured with mosquito C6/36 cells. Severe cytopathic effect was observed at day 3 p.i.. RT-PCR of the C6/36 cell lysate using dengue virus NS1 protein specific primers confirmed the existence of dengue viruses. And using restriction enzyme (BstEII) digestion to turn out the dengue viruses of mice brain extraction was derived from dengue-2 virus infectious cDNA clone. Although the virus titer (2.02×109 PFU/g) from dengue RNA transfected mice brain was higher then transfected C6/36, BHK and LLC-MK2 cells lines. In conclusion, our study demonstrates a suckling mice system to rapidly screen neurovirulence and produce infectious dengue viruses, which will be very useful for dengue virus study at molecular level.
|
