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研究生:劉亭勻
研究生(外文):TingYun Liu
論文名稱:二苯胺對人類T細胞之細胞毒性分析暨其對應之單鏈抗體基因庫之建構
論文名稱(外文):The Cytotoxicity of Diphenylamine in Human T Lymphocytes and the Construction of Its scFv Antibodies Library
指導教授:高振益高振益引用關係
指導教授(外文):JungYie Kao
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:英文
論文頁數:59
中文關鍵詞:二苯胺單鏈抗體基因庫細胞毒性分析噬菌體展示系統細胞凋亡細胞壞死非固醇類抗炎藥
外文關鍵詞:DiphenylaminescFv Antibodies LibraryCytotoxicityphage display systemapoptosisnecrosisNonsteroidal Anti-inflammatory Drugs
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二苯胺 ( Diphenylamine ) 是一種抗氧化劑,它的化學結構是非固醇類抗炎藥 ( Nonsteroidal Anti-inflammatory Drugs, NSAIDs ) 的骨架結構。在農業上用以處理蘋果表面以防止蘋果產生燙傷的現象 ( scald development ),並且也被高濃度的使用於染髮劑或殺蟲劑之中,因此二苯胺極可能因為農藥殘留問題而污染自然環境或因人體接觸造成生理上諸多方面的毒性傷害。已知二苯胺會抑制血小板的凝血功能,並具有肝毒性、腎毒性,最近研究報告指出它會輕微地增加人類淋巴球細胞之染色體複製時姐妹染色單體互換頻率。因此,本論文的主要研究目的除了更進一步闡明二苯胺對人類T淋巴球的細胞毒性,並且以噬菌體展示系統 ( phage display system ) 製備它的單鏈抗體基因庫 ( scFv antibodies library ) , 以期能夠利用抗體中和抗原的特性於未來應用在環境毒物的預防與治療。
首先我們以MTT assay探討二苯胺是否影響MOLT4細胞的正常生長速率, 結果發現二苯胺對T 細胞之生長速率的抑制作用呈現處理濃度依賴性 ( Dose-dependent ) 及處理時間依賴性 ( Time-dependent ),同時於LDH assay分析中發現高濃度的二苯胺會引起大量細胞溶解造成細胞壞死現象 ( Necrosis )。進一步以Cell Death Detectionplus Kit分析發現低濃度的二苯胺會造成T細胞的DNA斷裂而引起細胞凋亡 ( Apoptosis );另以SYBR Green I染細胞核,觀察發現有細胞核濃縮的現象。再者,萃取處理過二苯胺之人類T細胞的染色體DNA,觀察到電泳膠上有DNA ladder的情形。由以上結果發現高濃度的二苯胺會引發大量的T細胞走向細胞壞死,而在低濃度下二苯胺則會引起人類T細胞走向細胞凋亡。
另外我們亦製備二苯胺抗原經免疫小鼠後,萃取小鼠脾臟的RNA續以進行RT-PCR來產生各種不同的VH和VL 基因,建構針對二苯胺的單鏈抗體基因庫 ( scFv antibodies library )。

Diphenylamine is an antioxidant and has the skeleton structure of nonsteroidal anti-inflammatory Drugs. The apples were dipped in a solution of diphenylamine after harvest to prevent scald development. Diphenylamine was also found in the hair color dye and the pesticides in high concentration. It has been reported that diphenylamine alter platelet function, and has hepatotoxicity and nephrotoxicity. Recent advance report that diphenylamine produces a slight increase in sister chromatid exchange frequency in cultured human lymphocytes. In the present study, we investigate the cytotoxicity of diphenylamine in T lymphocytes, and prepare its scFv antibodies library by phage display system. First, we used the MTT assay to evaluate the effect of diphenylamine on proliferation rate of MOLT4 cell, and find that diphenylamine reduce the cell proliferation rate of T lymphocytes ( up to 60 % at the concentration of 200 M ). By LDH assay, T lymphocytes were lysed at high diphenylamine concentration. Furthermore, we test the effect of the low concentration of diphenylamine by Cell Death Detectionplus kit, and then find that the low diphenylamine concentration induce cell apoptosis undergo DNA fragmentation. By SYBR green I nuclear staining, diphenylamine induce nuclear condensation, and DNA fragmentation was visualized by agarose gel electrophoresis. These results indicated that diphenylamine at high concentration induced cell necrosis, and at low concentration induced cell apoptosis. At the same time, we synthesize the antigen of diphenylamine and then immunize BALB/c mouse for the production of anti-diphenylamine antibodies. After 2 mouths , we extract RNA from spleen cell and amplify the VH and VL genes by RT-PCR, and finally construct the anti-diphenylamine scFv antibodies library.

中 文 摘 要 …………………………………… i
英 文 摘 要 …………………………………… iii
緒 論 …………………………………… 1
實 驗 流 程 …………………………………… 10
材 料 與 方 法 …………………………………… 12
結 果 …………………………………… 29
討 論 …………………………………… 35
圖 表 …………………………………… 41
參 考 文 獻 …………………………………… 50

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