臺灣博碩士論文加值系統

本篇論文使用兩種中藥湯劑，麻杏石甘湯與荊防敗毒散研究抗流感病毒的機制。此兩種湯劑傳統上已知可用來治療感冒，發燒和頭痛，但是目前卻沒有此兩種湯劑抑制流感病毒的機制相關研究。在此，我們初步先使用中和試驗去驗證麻杏石甘湯與荊防敗毒散抗流感病毒A/WSN/33的能力。利用其他試驗進步探討藥物抑制病毒感染機制，如神經胺酸酶活性試驗，血液凝集抑制試驗以及PI3K/AKT 磷酸化試驗可偵測藥物是否可抑制流感病毒進入細胞。最後利用原子力顯微鏡可觀察被藥物處理過後病毒表面的結構是否有變化。在時程試驗中發現麻杏石甘湯除了抑制病毒進入細胞外，也會抑制病毒蛋白與RNA的合成。在原子力顯微鏡觀察下，可發現麻杏石甘湯會破壞病毒的表面。此外，也發現了麻杏石甘湯會抑制在病毒感染早期所誘發的AKT 磷酸化。以上這些科學證據也顯示了麻杏石甘湯對於治療流感的機制。與麻杏石甘湯不同的是荊防敗毒散在時程試驗中顯示了此湯劑抑制流感病毒的複製機制是在後期，如病毒的釋出或是病毒醣蛋白的運送，並不會干擾病毒蛋白與RNA的合成。在病毒斑減低試驗中發現，荊防敗毒散會減少病毒斑的大小與數量。最後經由免疫螢光染色確認病毒的醣蛋白血液凝集素的運送會被荊防敗毒散所阻止。因此，推測荊防敗毒散抗病毒複製的機制有可能是經由抑制病毒的聚集組裝，來達到抑制後代病毒的釋出。綜合以上實驗結果，證實麻杏石甘湯與荊防敗毒散與臨床用藥(例如：克流感)相同，都可用來治療季節性流感。

We investigated anti-influenza mechanism of two Chinese herbal decoctions: Ma-xing-shi-gan-tang (MXSGT, aka maxing shigan powder) and Ching-fang-pai-tu-san (CFPTS). MXSGT and CFPTS were traditionally used as a cure for the common cold, fever and headache. However, no studies have investigated the mode of action of MXSGT and CFPTS against influenza virus infection. The antiviral activity of nontoxic concentrations of MXSGT and CFPTS against influenza virus A/WSN/33 were examined by assaying inhibition of the virus-induced cytopathic effects (neutralization assay). The mode of MXSGT and CFPTS action were first examined with a time-of-addition assay of synchronized infections, followed by viral attachment and penetration assays. We also performed assays related to the inhibition of viral entry, such as neuraminidase (NA) activity, hemagglutinin (HA) activity, and phosphoinositide-3-kinase (PI3K)/AKT phosphorylation assays. The surface ultrastructure of the MXSGT or CFPTS-treated virus was revealed by atomic force microscopy. The synthesis of both viral RNA and protein was profoundly inhibited when the cells were treated with MXSGT. The time-of-addition assay demonstrated that MXSGT blocks the virus entry phase. High-resolution images and quantitative measurements made with atomic force microscopy confirmed that the viral surface structure was disrupted by MXSGT. We also established that viral entry, regulated by the PI3K/AKT signaling pathway, was abolished by MXSGT. These results give scientific support to the use of MXSGT in the treatment of influenza virus infections. CFPTS did not suppress viral RNA or protein synthesis. According to a time-of-addition assay, the antiviral mechanism of CFPTS may involve viral budding or viral glycoprotein exocytosis. A plaque reduction assay showed that CFPTS reduced both the plaque size and plaque quantity. The secretion of viral glycoprotein hemagglutinin was blocked by CFPTS by immunofluorescence microscopic analysis.
Therefore, the antiviral mechanism of CFPTS may inhibit the assembly of progeny virions and their subsequent release. Collectively, my studies suggest that MXSGT and CFPTS have potential utility in the management of seasonal pandemics of influenza virus infections, like other clinically available drugs, for example Tamiflu.

論文指導教授推薦書……………………………………………………i
論文口試委員審定書……………………………………………………ii
長庚大學博碩士論文著作授權書……………………………………………………iii
誌謝……………………………………………………iv
Abbreviation index……………………………………………………v
中文摘要……………………………………………………vii
Abstract……………………………………………………ix
Table and contents ……………………………………………………xi
Chapter I Introduction……………………………………………………1
1.1 Epidemiology of influenza virus……………………………………………………1
1.2 Background of influenza virus……………………………………………………1
1.3 Replication of influenza virus……………………………………………………3
1.4 Antiviral inhibitors against influenza virus……………………………………………………4
1.5 Chinese herb medicines：MXSGT and CFPTS……………………………………………………6
Chapter II Materials and methods……………………………………………………8
2.1 Cell culture and viruses……………………………………………………8
2.2 Preparation of MXSGT and CFPTS extracts……………………………………………………8
2.3 EC50 assay……………………………………………………9
2.4 Cytotoxicity MTT assay……………………………………………………10
2.5 Observation of CPE with microscopy……………………………………………………10
2.6 RNA extraction and quantitative reverse transcription–PCR (qRT–PCR)…………………………………………11
2.7 Detection of viral protein synthesis in the presence of MXSGT or CFPTS by immunoblotting……………………………………………………11
2.8 Indirect immunofluorescence assay……………………………………………………13
2.9 Time-of-addition assay……………………………………………………13
2.10 Hemagglutination assay (HA) and hemagglutination inhibition (HI)assay…………………………………14
2.11 NA assay……………………………………………………14
2.12 Attachment assay……………………………………………………15
2.13 Penetration assay……………………………………………………15
2.14 Plaque reduction assay……………………………………………………16
2.15 Atomic force microscopy (AFM)……………………………………………………16
Chapter III Experiment results……………………………………………………18
3.1 Results of MXSGT……………………………………………………18
3.1.1 Antiviral activity of MXSGT in MDCK cells……………………………………………………18
3.1.2 MXSGT impairs viral RNA and protein synthesis……………………………………………………19
3.1.3 Inhibitory mechanism determined by a time-of-addition assay…………………………………………………20
3.1.4 MXSGT inhibits viral entry……………………………………………………20
3.1.5 Binding of MXSGT to the viral surface to prevent viral penetration………………………………23
3.2 Results of CFPTS……………………………………………………24
3.2.1 Antiviral effectiveness and cytotoxicity of CFPTS……………………………………………………24
3.2.2 The effect of CFPTS on the synthesis of viral proteins or viral RNA……………………………25
3.2.3 Inhibitory mechanism of CFPTS studied with a time-of-addition assay……………………………25
3.2.4 CFPTS inhibits the secretion of viral glycoproteins……………………………………………………26
Chapter IV Discussion……………………………………………………29
4.1 Discussion of MXSGT……………………………………………………29
4.2 Discussion of CFPTS……………………………………………………33
Figures and tables……………………………………………………37
References……………………………………………………75

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