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研究生: 王康馨
Kang-Sing Wang
論文名稱: 巨噬細胞分泌之漿細胞瘤抑制因子誘發細胞自戕之現象
Plasmacytoma inhibitory factors secreted by marcophage induced
指導教授: 曾哲明
Tseng, Jer-Ming
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
畢業學年度: 82
語文別: 中文
論文頁數: 45
中文關鍵詞: 巨噬細胞;漿細胞;細胞自戕;胞殺作用;漿細胞瘤抑制素
英文關鍵詞: macrophage ; plasmacytoma cell ; apoptosis ; cytotoxic effect ;
論文種類: 學術論文
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  • 巨噬細胞株 P388D1 可分泌抑制因子抑制漿細胞瘤細胞株 MOPC-315
    的生長並造成胞殺作用,此抑制因子並非已發表在文獻上由巨噬細胞所分
    泌的胞殺因子,如 neutral protease、interleukin-1、interleukin-6、
    tumor necrosis factor 及 arginase 等。本實驗將濃縮之P388D1 培養基
    上清液,以 DEAE-Sephacel 離子交換色層分析法得到五群帶負電量不同的
    蛋白,其中 peak 4 與 peak 5 所含之蛋白具抑制 MOPC-315 生長及 IgA
    分泌之活性。將此五群蛋白經過 SDS-PAGE 電泳分析後,Peak 4 含分子量
    112-KDa 與 18-KDa 等兩種原先報導經過 S-300 Sephacryl chromatograt
    hy分析所得之漿細胞瘤抑制素,整個 peak 4所含抑制素的純化倍率為 181
    倍,而 peak 5 中之小分子蛋白可能是組成高分子量抑制因子的單體。本
    研究進一步證明漿細胞瘤抑制因子經由「細胞自戕作用」造成 MOPC-315細
    胞死亡。細胞自戕開始時細胞核內的核酸內切酵素開始活化,並將 DNA 切
    成 180-200 bp 或其倍數的片段,以 DNA agarose gel 電泳分析呈現階梯
    狀的螢光帶。且光帶量隨著 P388D1 上清液含量之增加而增加。此外,以
    Burton 之 diphenylamine 測試法發現隨著 P388D1上清液的含量增加,漿
    細胞瘤細胞株 MOPC-315 的 DNA 片段量亦隨著增加。 DNA 的片段量自 24
    小時後開始增加,至 72 小時達到最高。經過 DEAE-Sephacel 離子交換色
    層分析法所得到peak 4 及 peak 5 亦可造成 MOPC-315 DNA 片段量增加,
    且抑制活性高的分液使 MOPC-315 產生的 DNA 片段的量亦增加。

    Inhibitory factors secreted by P388D1 macrophage-like cell
    line exerted both inhibitory effect and cytotoxic effect on MOPC-
    315 plasmacytoma. The inhibitory factors were distinctfrom the de
    fined cytotoxic factors, such as neutral protease,interleukin-1,
    interleukin-6, tumor necrosis factor andarginase. In this study,
    the inhibitory factors in P388D1 supernatant were purified using
    DEAE-Sephacel ion-exchange chromatography. The major proteins
    were separated into five groups. Proteins in peak 4 and peak 5
    showed inhibitory effect on the growth and IgA secretion of MOPC-
    315. SDS-PAGE analysis of the five groups of proteins showed that
    peak 4 contained 102.3-KDa and 17.9-KDa proteins which had been
    observed by others using Sephacryl S-300 chromatography analysis
    in previous report. Peak 5 consisted of a low molecular weight pr
    otein which may be the monomer component of high molecular weight
    inhibitory factor reported by other. Furthermore, we demonstrated
    that plasmacytoma inhibitory factors induced the programmed cell
    death (apoptosis) of MOPC-315 cell. In the early stage of apoptos
    is,the endonucleases were activated and the DNA molecules were cl
    eaved into fragments that showed mutiples of 180-200 base pairs.
    DNA fragments could be visulized on gel electrophoresis and the
    amount of fragmented DNA was dose dependent. The DPA reaction mod
    ified from Burton provided an additional evidence ofapoptosis.
    The result of DPA assay showed that P388D1 supernatant generated
    DNA fragmentation in adose-dependentmanner and the amount of DNA
    fragments reached aplateau at 72 hours. Peak 4 and peak 5 obtain-
    ing from DEAE-Sephacel also increase the amount of DNA fragments.
    In addition, the fractions with higher inhibition activity genera
    ted more DNA fragmentation.
    Inhibitory factors secreted by P388D1 macrophage-like cell

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