- 學位論文
漿細胞瘤胞殺素誘導老鼠B淋巴球產生細胞自戕現象---- 正常細胞與腫瘤細胞的比較
Induction of apoptosis in murine B lymphocytes by a plasmacytoma cytotoxic factor:comparison between normal and tumor cells
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摘要
巨噬細胞對腫瘤細胞胞殺作用機制包括細胞與細胞間之直接接觸及分泌胞殺介質。本實驗室已確認，老鼠巨噬細胞P388D1細胞株培養基上清液中，含有胞殺因子PCF。PCF對MOPC-315老鼠漿細胞瘤細胞株具有胞殺作用，透過細胞自戕殺死腫瘤細胞。本研究進一步探討P388D1細胞株培養基上清液中是否還有別的因子，對其它腫瘤細胞或正常淋巴球細胞具有胞殺效果。先以0-20%（Fraction Ⅰ）、20-40%（Fraction Ⅱ）、40-60%（Fraction Ⅲ）飽和度硫酸銨鹽析出蛋白質成分，對MPC-11、MOPC-315及正常脾臟淋巴球作MTT胞殺作用檢測。結果顯示Fraction Ⅰ對正常脾臟淋巴球的胞殺作用最好，對MPC-11次之。Fraction Ⅱ也是對正常脾臟淋巴球的胞殺作用最好，對MPC-11次之。但Fraction Ⅲ對MOPC-315胞殺作用最好，對正常脾臟淋巴球次之。 進一步以Fraction Ⅰ、Fraction Ⅱ及Fraction Ⅲ對MPC-11、MOPC-315及正常脾臟淋巴球作細胞自戕檢測。以TUNEL及Annexin V試驗探討不同Fractions對MPC-11、MOPC-315及正常脾臟淋巴球DNA片斷化及磷酯酉先絲胺酸移位的影響。結果顯示Fraction Ⅰ會誘導正常脾臟淋巴球發生DNA片斷化及磷酯酉先絲胺酸(phosphatidylserine)移位。Fraction Ⅱ會誘導MPC-11發生DNA片斷化及磷酯酉先絲胺酸移位。Fraction Ⅲ會誘導MOPC-315發生DNA片斷化及磷酯酉先絲胺酸移位等一般細胞自戕所具有的現象。 TNF-α僅能造成L929發生細胞自戕，卻對正常脾臟淋巴球、MPC-11及MOPC-315沒有影響。這些細胞自戕現象亦能不被anti-TNF-α所中和，這個結果進一步排除TNF-α參與細胞自戕作用的可能性。單株抗體CB7.C2不能中和Fraction Ⅱ誘導MPC-11發生細胞自戕，進一步證實參與MPC-11細胞自戕的因子不是PCF。 綜合以上結果，不同因子分別誘導正常脾臟淋巴球、MPC-11及MOPC-315，發生細胞自戕。這些細胞自戕現象並非由TNF-α所引起。
並列摘要
The tumoricidal mechanisms of macrophages include cell-cell contact and secretion of cytotoxic agents.Our previous report indicated that the culture supernatant of P388D1 murine macrophage-like cell line was cytotoxic to MOPC-315 plasmacytoma.One of the cytotoxic factors in culture supernatant , which was designated PCF was characterized.PCF killed the tumor cells by induction of apoptosis. This study further identified other factors in P388D1 culture supernatant which might be cytotoxic to other tumor cells or normal lymphocytes. Protein components in P388D1 culture supernatnat were fractionated by 0-20% (Ⅰ) ,20-40% (Ⅱ) and 40-60% (Ⅲ) saturation of ammonium sulfate. Each fraction was assayed for its cytotoxic activity against MPC-11 plasmacytoma , MOPC-315 plasmacytoma and normal spleen lymphocytes by MTT method. Results suggested that Fraction Ⅰ was cytotoxic to the normal lymphocytes. The same fraction had less cytotoxic to MPC-11 cells. Fraction Ⅱ was also cytotoxic to normal cells but less extent to MPC-11 cells.Fraction Ⅲ showed predominant cytotoxicity against MOPC-315 , and less extent to normal lymphocytes and MPC-11 cells. To study further the possible involvement of apoptosis during the process of cytotoxicity , the TUNEL assay was used to monitor the generation of DNA fragmentation.Results suggested that Fraction Ⅰ induced DNA fragmentation in normal lymphocytes , Fraction Ⅱ predominantly induced DNA fragmentation in MPC-11 cells , and Fraction Ⅲ induced DNA fragmentation in MOPC-315 cells. Induction of apoptosis in three types of target cells was further confirmed by detection of phosphatidylserine translocation by Annexin V assay. TNF-α alone induced apoptosis in L929 cells but had no effect on MPC-11, MOPC-315, and normal lymphocytes.In addition , an anti-TNF-α antibody failed to neutralize the cytotoxicity of P388D1 culture supernatant against three types of targets cells.An anti-PCF antibody CB7.C2 failed to neutralized fraction Ⅱ- induced apoptosis in MPC-11 cells , suggesting that cytotoxic factor against MPC-11 in fraction Ⅱ is distinct from TNF-α or the PCF characterized by previous papers.
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