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研究生(外文):TingYun Liu
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(外文):JungYie Kao
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

1.Johnson GD, Geronimo J and Hughes DL (1997) Diphenylamine residues in apples ( Malus domestica Borkh.), cider, and pomace following commercial controlled atmosphere storage. J Agric Food Chem 45: 976-979.
2. Mir NA and Beaudry R (1999) Effect of superficial scald suppression by diphenylamine application on volatile evolution by stored Cortland apple fruit. J Agric Food Chem 47: 7-11.
3. Ju Z and Bramlage WJ (1999) Phenolics and lipid-soluble antioxidants in fruit cuticle of apples and their antioxidant activities in model systems. Phostharvest Biol. and Technol. 16: 107-118.
4. Kralj M, Kapitanovic S, Kovacevic D, Lukac J, Spaventi S and Pavelic K (2001) Effect of the nonsteroidal anti-inflammatory drug indomethacin on proliferation and apoptosis of colon carcinoma cell. J Cancer Res Clin Oncol.127: 173-179.
5. Rao GHR, Tate MR, Murthy M, Hebbel RP and White JG (1994) Influence of antioxidants on arachidonic acid metabolism and platelet function. Biochem Med and Met Bio 51: 74-79.
6. Pasquini R, Scassellati-Sforzolini G, Dolara P, Pampanella L, Villarini M, Caderni G, Fazi M and Fatigoni C (1994) Assay of linuron and a pesticide mixture commonly found in the Italian diet, for promoting activity in rat liver carcinogenesis. Pharmacol Toxicol 75: 170-176.
7. Masubuchi Y, Saito H and Horie T (1998 ) Structural requirements for the hepatotoxicity of nonsteroidal anti-inflammatory drugs in isolated rat hepatocytes. J Pharmacol Exp Ther 287: 208-213.
8. Masubuchi Y, Yamada S and Horie T (1999) Diphenylamine as an important structure of nonsteroidal anti-inflammatory drugs to uncouple mitochondrial oxidative phosphorylation. Biochem Pharmacol 58: 861-865.
9. Masubuchi Y, Yamada S and Horie T ( 2000 ) Possible mechanism of hepatocyte injury induced by diphenylamine and its structurally related nonsteroidal anti-inflammatory drugs. J Pharmacol Exp Ther 292: 982-987.
10.Lodovici M, Casalini C, Briani C and Dolara P (1997) Oxidative liver DNA damage in rats treated with pesticide mixtures. Toxicology 117: 55-60.
11.Lenz SD, Carlton WW (1990) Diphenylamine-induced renal papillary necrosis and necrosis of the pars recta in laboratory rodents. Vet Pathol 27: 171-178.
12. Lenz SD and Carlton WW (1991) Decreased incidence of diphenylamine-induced renal papillary necrosis in Syrian hamsters given dimethylsulphoxide. Food Chem Toxicol 29: 409-418.
13. Sorrenyino F, Fella A and Pota A (1978) Diphenylamine-induced renal lesions in the chicken. Urol Res 6: 71-75
14. Yoshida J, Shimoji N, Furuta K, Takamura N, Uneyama C,Yazawa R, Imaida K and Hayashi Y (1989) Twenty-eight day repeated dose toxicity testing of diphenylamine in F344 rats. Eisei Shikenjo Hokoku 107: 56-61.
15. Dolara P, Torricelli F, Antonelli N (1994) Cytogenetic effect on human lymphocytes of a mixture of fifteen pesticides commonly used in Italy. Mut Res 325: 47-53.
16. Ardito G,Bramanti B, Bigatti P, Lamberti L and Dolara P (1996) Cytogenetic effect of thiabendazole and diphenylamine on cultured human lymphocytes: sister chromatid exchanges and cell cycle delay. Boll Soc Ital Biol Sper 72: 171-178.
17. Huston JS, Levinson D, Mudgett-Hunter M, Tai MS, Novotny J, Margolies MN, Ridge RJ, Bruccoleri RE, Haber E, Crea R, et al.(1988) Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc. Natl. Acad. Sci. USA 85: 5879-5883
18. McCafferty J, Griffiths AD, Winter G, Chiswell DJ. (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348: 552-554.
19. Haidaris CG, Malone J, Sherrill LA, Bliss JM, Gaspari AA, Insel RA, Sullivan MA. (2001) Recombinant human antibody single chain variable fragments reactive with Candida albicans surface antigens. J Immunol Methods 257: 185-202.
20. Amersdorfer P, Wong C, Chen S, Smith T, Deshpande S, Sheridan R, Finnern R, Marks JD. (1997) Molecular characterization of murine humoral immune response to botulinum neurotoxin type A binding domain as assessed by using phage antibody libraries. Infect Immun, 65: 3743-3752.
21. Tse E, Lobato MN, Forster A, Tanaka T, Chung GT, Rabbitts TH. (2002) Intracellular antibody capture technology: application to selection of intracellular antibodies recognising the BCR-ABL oncogenic protein. J Mol Biol 317: 85-94.
22. Foy BD, Killeen GF, Frohn RH, Impoinvil D, Williams A, Beier JC. (2002) Characterization of a unique human single-chain antibody isolated by phage-display selection on membrane-bound mosquito midgut antigens. J Immunol Methods 261: 73-83.
23. Goletz S, Christensen PA, Kristensen P, Blohm D, Tomlinson I, Winter G, Karsten U. (2002) Selection of large diversities of antiidiotypic antibody fragments by phage display. J Mol Biol 315: 1087-1097.
24. Schier R, McCall A, Adams GP, Marshall KW, Merritt H, Yim M, Crawford RS, Weiner LM, Marks C, Marks JD. (1996) Isolation of picomolar affinity anti-c-erbB-2 single-chain Fv by molecular evolution of the complementarity determining regions in the center of the antibody binding site. J Mol Bio 263: 551-567.
25. Daugherty PS, Chen G, Iverson BL, Georgiou G. (2000) Quantitative analysis of the effect of the mutation frequency on the affinity maturation of single chain Fv antibodies. Proc Natl Acad Sci U S A 97: 2029-2034.
26. Chowdhury PS, Pastan I. (1999) Improving antibody affinity by mimicking somatic hypermutation in vitro. Nat Biotechnol 17: 568-572.
27. Vitaliti A, Wittmer M, Steiner R, Wyder L, Neri D, Klemenz R. (2000) Inhibition of tumor angiogenesis by a single-chain antibody directed against vascular endothelial growth factor. Cancer Res 60: 4311-4314.
28. Poul MA, Becerril B, Nielsen UB, Morisson P, Marks JD. ( 2000 )
Selection of tumor-specific internalizing human antibodies from phage libraries. J Mol Biol 301: 1149-1161.
29. Bera TK, Kennedy PE, Berger EA, Barbas CF 3rd, Pastan I. (1998) Specific killing of HIV-infected lymphocytes by a recombinant immunotoxin directed against the HIV-1 envelope glycoprotein. Mol Med 4: 384-391.
30.DeNardo SJ, DeNardo GL, DeNardo DG, Xiong CY, Shi XB, Winthrop MD, Kroger LA, Carter P. (1999) Antibody phage libraries for the next generation of tumor targeting radioimmunotherapeutics. Clin Cancer Res 5: 3213s-3218s.
31.Bidere N and Senik A (2001) Caspase-independent apoptotic pathways in T lymphocytes: a minireview. Apoptosis 6: 371-375.
32.Di Baldassarre A, Secchiero P, Grilli A, celeghini C, Falciere E and Zauli G (2000) Morphological features of apoptosis in hematopoietic cells belonging to the T-lymphoid and myeloid lineages. Cell Mol Biol 46: 153-161
33. Aravind L, Dixit VM and KooninEV (2001) Apoptotic molecular machinery: vastly increased complexity in vertebrates revealed by genome comparisons. Science 291: 1279-1284.
34.Martelli AM, Zweyer M, Ochs RL, Tazzari PL, Tabellini G, Narducci P and Bortul Roberta (2001) Nuclear apoptotic changes: an overview. J Cell Biochem 82: 634-646.
35. Chan SL, Mattson MP, (1999) Caspase and calpain substrate: roles in synaptic plasticity and cell death, J Neurosci Res, 58: 167-190.
36. Putcha GV, Deshmukh M, Johnson EM, (1999) Bax translocation is a critical event in neuronal apoptosis: regulation by neuroprotectants, Bcl-2, and caspase, J Neurosci, 19: 7476-7485.
37. Nakayama M, Neri D, Ohara O. (2001) A new simplified method for preparation of a synthetic phage antibody with practically acceptable detection sensitivity on immunoblots. Hum Antibodies 10: 55-65.
38.Mossner E, Koch H, Pluckthun A. (2001) Fast selection of antibodies without antigen purification: adaptation of the protein fragment complementation assay to select antigen-antibody pairs.
J Mol Biol 308: 115-122.
39. Neame SL, Rubin LL, Philpott KL, (1998) Blocking cytochrome c activity within intact neurons inhibits apoptosis, J Cell Biol, 142:1583-1593.
40. Kala M, Bajaj K, Sinha S. (2001) Direct antigen capture by soluble scFv antibodies. A method for detection, characterization, and determination of affinity. Appl Biochem Biotechnol 90:11-22.
41. Eeckhout D, Fiers E, Sienaert R, Snoeck V, Depicker A, De Jaeger G. (2000) Isolation and characterization of recombinant antibody fragments against CDC2a from Arabidopsis thaliana. Eur J Biochem 267: 6775-6783.
42. Boel E, Verlaan S, Poppelier MJ, Westerdaal NA, Van Strijp JA, Logtenberg T. (2000) Functional human monoclonal antibodies of all isotypes constructed from phage display library-derived single-chain Fv antibody fragments. J Immunol Methods 239: 153-166.
43. Klimka A, Matthey B, Roovers RC, Barth S, Arends JW, Engert A, Hoogenboom HR. (2000) Human anti-CD30 recombinant antibodies by guided phage antibody selection using cell panning. Br J Cancer 83: 252-260.
44. Amersdorfer P, Marks JD. (2000) Phage libraries for generation of anti-botulinum scFv antibodies. Methods Mol Biol 145: 219-240.
45.Krebs B, Rauchenberger R, Reiffert S, Rothe C, Tesar M, Thomassen E, Cao M, Dreier T, Fischer D, Hoss A, Inge L, Knappik A, Marget M, Pack P, Meng XQ, Schier R, Sohlemann P, Winter J, Wolle J, Kretzschmar T. (2001) High-throughput generation and engineering of recombinant human antibodies. J Immunol Methods 254: 67-84.
46. Winthrop MD, Denardo GL, Denardo SJ. (2000) Antibody phage display applications for nuclear medicine imaging and therapy. Q J Nucl Med 44: 284-295.
47. Williamson P, Matthews R. (1999) Development of neutralising human recombinant antibodies to pertussis toxin. FEMS Immunol Med Microbiol 23 : 313-9.
48. Carmichael J, DeGraff WG, Gazdar AF, Minna JD and Mitchell JB (1987) Evaluationof a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res 47: 936-942.
49. Decker T and Lohmann-Matthes ML (1988) A quick and simple method for the quantitation of lactate dehydrogenase release in measurement of cellular cytotoxicity and tumor necrosis factor (TNF) activity. J Immunol Meth 15: 61-69.
50. Herrmann M, Lorenz HM, Voll R, Grunke M, Woith W and Kalden JR (1994) A rapid and simple method for the isolation of apoptotic DNA fragments. Nuclei Acid Research 22: 5506-5507.

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