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題 名 | 漿細胞瘤抑制素誘發細胞自戕之研究=Plasmacytoma Cytotoxic Factor Induce Apoptosis in Tumor Cell |
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作 者 | 劉倩君; | 書刊名 | 康寧學報 |
卷 期 | 1:2 1999.03[民88.03] |
頁 次 | 頁161-186 |
分類號 | 415.138 |
關鍵詞 | 漿細胞瘤抑制素; 細胞自戕; IL-1β轉換酵素; Plasmacytoma cytotoxic factor; Apoptosis; ICE; |
語 文 | 中文(Chinese) |
中文摘要 | 巨噬細胞的生物效應之一,即對腫瘤細胞產生胞殺作用。本實驗室數年前發現一種 對MOPC-315漿細胞瘤具有胞殺作用的因子(Plasm-ytoma cytotoxic factor;PCF),對 MOPC-315、MPC-11漿細胞瘤及FO等骨髓瘤細胞有毒性,但對J558骨髓瘤細胞沒有影響。本實 驗以DEAE-Sephacel離子交換色層管柱,由P388Dl培養基上清液純化PCF,再以此PCF為免疫原 製備一系列單株抗體,這些單株抗體皆可中和P388Dl對MOPC315細胞的胞殺作用。以硫胺酸分 割法(40-60% 飽和度)初步純化PCF之後,直接透過抗PCF之單株抗體(CB7-C2)製成之免疫親和 性管柱純化出PCF,在SDS-PAGE分析中呈一M.W.62KDa之蛋白帶,以CB7作西方點墨法,證實此 62KDa蛋白為PCF,但是以CB7-C2為探針則呈現一分子量約130 KDa之蛋白帶,顯示PCF可能以 雙倍體存在。PCF之N端胺基酸序列為Ala-lle-Leu-Leu-Ala,呈相對油溶性。初步研究結果顯 示,粗純化之PCF會造成MOPC-315 DNA斷裂現象,即PCF殺死腫瘤之機制可能是細抱自狀 (apoptosis)。以純化之PCF進一步處理MOPC-315細肥後,經agarose gel與TUNEL標示DNA片 段,以及以Annexin V標示phospatidylserine技術分析,證實DNA斷裂程度及apoptosis的程 度,與PCF之用量及處理之時間長度呈正比。而抗PCF單株抗體CB7可使apoptosis的程度降低 或完全消除,進一步證實造成MOPC-315 apoptosis現象的因子,確實是分子量62KDa的PCF。 經PCF處理過之細抱以RT-PCR技術分析,發現其IL-lβ轉換酵素(IL-1βconverting enzyme; ICE)之mRNA量顯著增加,加入CB7單株抗體即使ICE基因表現顯著降低,證明PCF透過與TNFα 相似的ICE pathway,造成MOPC-315腫瘤細胞之細胞自戕現象。 |
英文摘要 | Tumoricidal activity is one of the major effector functions of activated macrophages. Our previous study demonstrated that the culture supernatant of P388D1 murine macrophage-like cells showed a cytotoxic effect on plasmacytoma MOPC-3 15, MPC-11 and myeloma FO but no effect on J558 cells. In this study, the plasmacytoma cytotoxic factor (PCF) in P388D1 culture supernatant was partially purified by a DEAE-Sephacel ionic-exchanger chromatography and the partially purified PCF was used as an immunogen to prepare a panel of monoclonal antibodies against PCF. All monoclonal antibodies partially blocked the P388D1-mediated tumoricidal activity. A large scale purification was performed by ammonium sulfate fractionation (40-60% saturation), followed by an immunoaffinity chromatography using one of the anti-PCF monoclonal antibodies CB7-C2 (gamma-1/lambda, titer 6.5 x 10□.). The affinity-purified PCF had IC50 at 3.11 μg /ml for 2x10□ MOPC 315 cells and showed a major band with an estimated molecular weight of 62 KDa on SDS-PAGE gel. 087 did recognized the 62 KDa protein but CB7.C2 recognized a single band with an estimated Mr of 130 KDa on Western blotting, suggesting that the native form of PCF could be a dimer. The NH3-terminal sequence of PCF is currently analyzed. The first five amino acid, Ala-Ile-Leu-Leu-Ala, is relatively hydrophobic and is irrelevant to the sequence of well-documented murine macrophage-derived proteins. Our preliminary data suggested that the PCF might kill the plasmacytoma cells by PCF-induced apoptosis. In this study, the tumoe cells were treated with various dose of PCF and the extent of DNA fragmentation was monitored by both agarose gel electrophoresis and terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) method. Apoptosis was further confirmed by detection of phosphatidylserine redistribution by annexin V staining technique. All analytical data suggested that PCF induced apoptosis in MOPC 315 plasmacytoma cells with a dose-dependent and time-dependent manner. The purified PCF-induced apoptosis could be neutralized by anti-PCI antibody CB7. The extent of IL-1β converting enzyme (ICE) gene expression was estimated by RT-PCR. Result indicated that PCF induced a significant increase in ICE gene expression and this augmented effect could be significantly reduced by anti-PCF antibody CB7, suggesting that PCF might induce an apoptosis in MOPC 315 cells through ICE pathway. |
本系統中英文摘要資訊取自各篇刊載內容。