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研究生:張家緯
研究生(外文):Chia-Wei Chang
論文名稱:以電流感測法測量石斑魚細胞活性與應用於台灣石斑虹彩病毒感染檢測之可行性
論文名稱(外文):Application feasibility of amperometric measurement of grouper cell activity in detection of grouper iridovirus of Taiwan (TGIV) infection
指導教授:周信佑周信佑引用關係
指導教授(外文):Hsin-Yiu Chou
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:水產養殖學系
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:76
中文關鍵詞:電流式感測石斑泳鰾細胞耗氧
外文關鍵詞:amperometric sensorgrouper swim blabber celloxygen consumption
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首先以循環伏安法 ( Cyclic voltammetry ) 測量培養液中不同氧氣濃度下,單一氧進行還原作用的電壓位置,得到還原峰電壓位置在-0.4V~-0.5V,且確認氧濃度與所測得的電流呈現正比關係。之後將電壓固定於-0.4V,以電流感測法進行石斑魚泳鰾細胞 (SB cell) 的耗氧分析。結果細胞量多、耗氧量增高,培養基中殘留氧低則電流呈現下降的現象。進一步以同法偵測台灣石斑虹彩病毒 ( Grouper iridovirus of Taiwan, TGIV ) 感染對SB細胞耗氧的影響,結果M.O.I. (multiplicity of infection) 為1的組別,病毒接種1~4.5小時內的電流量持續上升,顯示感染細胞的耗氧量減少,而在7~10小時逐漸平穩,但是感染後15小時電流下降,顯示細胞耗氧又開始增加。此外,將TGIV以三個M.O.I.=1、0.1與0.01接種SB細胞,結果M.O.I.= 1組的平均電流量高於 M.O.I.= 0.1和0.01組別,且感染初期會造成細胞呼吸量的下降,而M.O.I.=0.1和0.01的組別電流量和控制組相同。相對觀察TGIV 感染SB 細胞的病變情形,直到感染三~四天後才開始出現細胞病變效應(cytopathic effect, CPE),顯示電流感測法與傳統接種細胞之病變觀察比較,能更早檢測病毒的感染。進一步將電流感測法應用於免疫激活物對抗病毒感染之評估,結果發現馬尾藻多醣、鹿角菜膠和干擾素能夠讓SB細胞受TGIV感染後產生細胞氧代謝降低的現象延緩,氧代謝降低的現象到感染後12~15小時才出現。而in vitro觀察下,馬尾藻多醣、鹿角菜膠和鯉魚干擾素均有刺激細胞產生抗病毒物質的能力,馬尾藻多醣和鹿角菜膠濃度為0.1 mg/ml時以及鯉魚干擾素稀釋50~1000倍之實驗組均能夠TGIV病毒力價下降 1~2個log TCID50/ml。以MTT 分析免疫激活物處理細胞之活性變化,結果並沒有無明顯改變,推測免疫激活物不會直接影響細胞活性,可能是透過誘發抗病毒物質的分泌來抑制病毒的感染。
Using cyclic voltammetry to measure the reductive potential at different oxygen concentrations, a reductive potential ranging from -0.4 to -0.5v was obtained. The results indicate that the cell density is relative to oxygen consumption, and the residual oxygen in culture medium is proven to be directly proportional to the responding current. We analyzed oxygen consumption of the SB cells infected with TGIV by cyclic voltammetry at -0.4v. When M.O.I. is 1, a continuous increase of the reduction current shows the decrease of the oxygen consumption of cells at 1~4.5 hour post-infection (hpi). The quantity of the oxygen consumption restores to a normal reduction current at 7~10 hpi. At 15 hpi, decrease of the reduction current shows the increase of the oxygen consumption. Further more, SB cell were infected with TGIV at M.O.I.=1、0.1 and 0.01.When M.O.I.=1, the average current was greater than current recorded of M.O.I.=0.1 and 0.01,and the cellular respiration decrease in initial infection. The average current at M.O.I.=0.1 and 0.01 is similar to the result of control. Oppositely, observe cytopathic effect (CPE) was observed until 3~4 days postinfection after TGIV infection. Compared with the traditional CPE observation, amperometric measurement can examine virus infection ealier. Further more, amperometric measurement was used to estimate immunostimulation inhibition for TGIV infection . We detect that sargassum、carrageenan and interferon can suspend the inhibition of total cellular respiration caused dy TGIV infection. This inhibition occured until 12~15 hour post-infection (hpi), indicating that sargassum、carrageenan and interferon can stimulate SB cell secretion antiviral substance.When sargassum and carrageenan concentration at 0.1 mg/ml,and the interferon from crap dilute 50~1000 fold, the titer of TGIV decrease 1~2 log TCID50/ml. MTT assay results indicate that there is no obvious cellular response variation after immuostimulant connect with SB cell. We suggest that immunostimulant would not increase cellular response,but inhibit virus infection directly.
目 錄
謝誌……………………………………………….…………………………i
中文摘要………………………………………………………………...….ii
英文摘要…………………………………………………………………...iv
目錄………………………………………………………...………………vi
圖目錄…………………………………………………………………...…xi
前言…………………………………………………………………………1
文獻整理……………………………………………………………………3
一、生醫感測器介紹……………………………………………………..3
二、動物病毒感染與細胞呼吸作用的影響……………………………...6
三、免疫激活物對細胞呼吸的影響……………………………………...9
四、虹彩病毒簡介………………………………………………………12
材料與方法………………………………………………………………..14
一、電流與氧濃度關係測定……………………………...……………14
(一) 實驗材料
1、溶氧濃度檢測器…………………………………………………..14
2、電流式生醫感測器………………………………………………..14
3、三電極系統………………………………………………………14
4、培養基配置………………………………………………………14
5、循環伏安法介紹…………………………………………………15
(二) 實驗方法
1、溶氧實驗架設……………………………………………………17
2、電化學測量……………………………………………………….17
二、電流感測法測量細胞基本耗氧反應………………………………17
(一) 實驗材料
1、細胞株……………………………………………………………..17
2、培養基…………………………………………………………….17
3、細胞分散劑 (0.1%胰蛋白?-EDTA 溶液)………………………18
(二) 實驗方法…………………………………………………………18
1、細胞培養…………………………………………………………18
2、電化學測量……………………………………………………….18
三、台灣石斑虹彩病毒感染後細胞耗氧反應偵測…………………….19
(一) 實驗材料
1、病毒……………………………………………………………….19
2、細胞活性測量藥劑……………………………………………….19
(二) 實驗方法
1、 TGIV 感染SB細胞………………………………………………19
2、 電化學測量………………………………………………………..20
3、 MTT 分析………………………………………………………...20
四、免疫激活物對細胞活性影響………………………………………20
(一) 實驗材料
1、藻類多醣………………………………………………………….20
2、干擾素……………………………………………………………..21
(二) 實驗方法
1、電化學測量…………………………………………………………21
2、MTT 分析…………………………………………………………21
五、免疫激活物對SB細胞抗TGIV之影響……………………….…21
1、細胞培養分析病毒力價…………………………………………21
(1) 藻類多醣硫酸根含量測試………………………………………21
(2) 細胞培養分析……………………………………………………22
A、免疫激活物對病毒中和效果……………………………………22
B、免疫激活物誘發細胞產生抗病毒物質…………………………23
C、免疫激活物抑制病毒增殖………………………………………23
2、電流感測法測試………………………………………………….23
3、MTT 分析………………………………………………………...23
結果…………………………………………………………………….....25
一、電化學與氧濃度關係測定………………………………………..25
二、細胞基本耗氧反應偵測…………………………………………..25
三、台灣石斑虹彩病毒感染後細胞耗氧反應偵測…………………..26
1、電化學測量………………………………………………………..26
2、MTT 分析…………………………………………………………26
四、免疫機活物對細胞活性影響偵測…………………………………26
1、電化學測量…………………………………………………………26
2、MTT 分析………………………………………………………….27
五、免疫激活物對SB細胞抗TGIV之影響………………………….27
1、細胞培養分析病毒力價……………………………………….27
(1) 藻類多醣體含硫酸根量測試……………………………………27
(2) 細胞培養分析……………………………………………………27
A、免疫激活物對病毒中和效果……………………………………27
B、免疫激活物誘發細胞產生抗病毒物質…………………………28
C、免疫激活物抑制病毒增殖………………………………………28
2、電流感測法測試……………………………………………………..28
3、 MTT 分析…………………………………………………………29
討論………………………………………………………………………..30
參考文獻…………………………………………………………………..36
圖…………………………………………………………………………..44
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