臺灣博碩士論文加值系統

近年來藉由節肢動物 (Arthropod)所傳播的黃質病毒感染病例有逐年升高的趨勢，且登革病毒和日本腦炎病毒等黃質病毒散佈的範圍很廣。登革熱和登革出血熱是因為登革病毒感染所引起的，目前仍是世界性的公共衛生問題。海藻中已知的成分－硫酸多醣(sulfated polysaccharides)，其生理活性被廣泛的應用於抗病毒、抗腫瘤、抗氧化、抗凝血劑、降膽固醇等療效。本研究主要是以細胞培養的方式，探討由綠藻屬之石蓴(Ulva lactuca)的三種水溶萃取物A1、A4及A8，是否能有效抑制黃質病毒感染細胞。100 µg/ml的三種石蓴萃取物並無細胞毒性；以間接免疫螢光法觀察，100 µg/ml之A4或A8對DEN2感染BHK-21細胞之抑制率約九成，而對HepG2細胞的感染亦有明顯的抑制。進一步研究發現石蓴萃取物抑制DEN2之作用機制是在病毒感染的初期，可能是抑制病毒與細胞之間的吸附及/或穿透步驟。另外，該萃取物對同屬於黃質病毒的JEV感染亦具抑制效果，但是其卻對α病毒屬(Alphavirus)的Sindbis virus感染BHK-21細胞無抑制作用(即使在250 µg/ml和500 µg/ml的高濃度下)。由以上結果顯示，某些石蓴水溶萃取物具有專一制黃質病毒感染之能力，而此能力是否與其所含硫酸多醣的種類及含量有關值得進一步研究。

Viral infectious diseases caused by arthropod-borne flaviviruses have been gradually increased worldwide in recent years, especially including dengue virus (DEN) and Japanese encephalitis virus (JEV) prevalent in Taiwan and other South Asia countries. Dengue viruses cause dengue fever and dengue related diseases that represent a global public health problem. Seaweeds have known to possess an ingredient of sulfated polysaccharides that have been widely used as curative agents for anti-virus, anti-tumor, anti-oxidation, anti-coagulant, and control of normal cholesterol levels. The main purpose of this thesis is to study, by using cell cultures, the anti-DEN effect of the water-soluble extracts from Ulva lactuca, which belongs to green seaweed. Three homogeneous extracts of Ulva lactuca leaves, A1, A4 and A8, were used in this study and we found them had no cytotoxicity to the cultured cells at as high as 100 µg/ml. By indirect immunofluorescence assay, we observed that 100 µg/ml of A4 or A8 could suppress DEN infection to BHK-21 cells by 90%, and similarly we found they could also inhibit DEN infection to human hepatocyte HepG2 cells. We further noticed that these extracts were active against DEN infection only during the early stage of virus adsorption and penetration. Moreover, another flavivirus JEV was also found to be blocked to certain degrees by Ulva lactuca extracts when infected to BHK-21 cells. However, Sindbis virus, a member of Alphaviruses, appeared to be not suppressed by these extracts even their concentrations were used as high as 500 µg/ml. Together, our results demonstrate that the extracts from green seaweed Ulva lactuca, comprising abundant sulfated polysaccharides, have a specific anti-flavivirus effect in cultured system and the medical values of these extracts are worth further studying.

目 錄
中文摘要........Ⅰ
Abstract........Ⅱ
目錄............Ⅲ
圖表目錄........Ⅴ
壹、前言........1
貳、文獻回顧....3
一、黃質病毒之簡介....3
二、黃質病毒基因結構 (Genome structure).....3
三、黃質病毒傳染途徑......4
四、海藻的成分及其生理活性.....6
參、實驗設計......8
肆、材料與方法....9
一、材料......9
(一)原料......9
(二)實驗所使用之細胞株....9
(三)實驗所使用之病毒株....9
(四)實驗所使用之抗體......9
(五)化學藥品與試劑.......10

二、實驗方法......10
(一)細胞株培養....10
(二)細胞存活率之分析 (XTT assay)....11
(三)DEN2之增殖....11
(四)JEV RP9之增殖...12
(五)病毒效價之測定(Virus titration).....12
(六)病毒感染(Virus infection).....13
(七)間接免疫螢光染色法(Indirect immunofluoresent assay)...13

伍、結果........15
一、石蓴萃取物對培養細胞之毒性..........15
二、石蓴萃取物對DEN2感染BHK-21細胞之影響......15
三、石蓴萃取物對不同宿主細胞感染DEN2後之影響....18
四、石蓴萃取物在不同時間點處理下對DEN2感染之影響....20
五、石蓴萃取物對病毒感染細胞初期之影響......21
六、石蓴萃取物對其他RNA病毒感染BHK-21細胞之影響....22
陸、討論....24
參考文獻....29
圖表........33
附錄........61

圖 表 目 錄
圖一、石蓴萃取物對BHK-21細胞毒性之影響......33
圖二、石蓴萃取物A1在不同濃度下於BHK-21細胞對DEN2感染後
24小時之影響......34
圖三、石蓴萃取物A4在不同濃度下於BHK-21細胞對DEN2感染後
24小時之影響......35
圖四、石蓴萃取物A8在不同濃度下於BHK-21細胞對DEN2感染後
24小時之影響......36
圖五、石蓴萃取物A1在不同濃度下於BHK-21細胞對DEN2感染後
48小時之影響......37
圖六、石蓴萃取物A4在不同濃度下於BHK-21細胞對DEN2感染後
48小時之影響......38
圖七、石蓴萃取物A8在不同濃度下於BHK-21細胞對DEN2感染後
48小時之影響......39
圖八、石蓴萃取物A1在不同濃度下於BHK-21細胞對DEN2感染後
72小時之影響......40
圖九、石蓴萃取物A4在不同濃度下於BHK-21細胞對DEN2感染後
72小時之影響......41
圖十、石蓴萃取物 A8在不同濃度下於BHK-21細胞對DEN2感染後
72小時之影響......42
圖十一、石蓴萃取物A4和A8於BHK-21細胞對DEN2感染24小時後以
plaque assay分析病毒效價......43
圖十二、石蓴萃取物於BHK-21細胞感染DEN2後24小時之影響....44
圖十三、石蓴萃取物對HepG2細胞毒性之影響......45
圖十四、石蓴萃取物A1在不同濃度下於HepG2 細胞對DEN2感染
後48小時之影響.......46
圖十五、石蓴萃取物A4在不同濃度下於HepG2細胞對DEN2感染
後48小時之影響.......47
圖十六、石蓴萃取物A8在不同濃度下於HepG2細胞對DEN2感染
後48小時之影響.......48
圖十七、在DEN2 感染前先加入石蓴萃取物A4對BHK-21細胞之影響......49
圖十八、在DEN2 感染的同時加入石蓴萃取物A4 對BHK-21細胞之影響......50
圖十九、在DEN2感染後加入石蓴萃取物A4對BHK-21細胞之影響......51
圖二十、在DEN2感染前先加入石蓴萃取物A8對BHK-21細胞之影響......52
圖二十一、在DEN2 感染的同時加入石蓴萃取物A8對BHK-21細胞之影響......53
圖二十二、在DEN2 感染後加入石蓴萃取物A8對BHK-21細胞之影響......54
圖二十三、石蓴萃取物A4對DEN2感染BHK-21細胞吸附之影響......55
圖二十四、石蓴萃取物A8對DEN2感染BHK-21細胞吸附之影響......56
圖二十五、石蓴萃取物A4對JEV感染BHK-21細胞之影響...........57
圖二十六、石蓴萃取物A8對JEV感染BHK-21細胞之影響...........58
圖二十七、石蓴萃取物對Sindbis感染BHK-21細胞之影響.........59
圖二十八、石蓴萃取物對Sindbis感染BHK-21細胞之影響.........60
附錄一 綠藻屬之石蓴(Ulva lactuca) 萃取物之萃取條件及成分..61

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