超抗原SEB激活诱导CD4＋T细胞凋亡模型的建立
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作者：熊世勤 朱锡华 黄云辉
单位：第三军医大学免疫学教研室，全军免疫学研究所，重庆 400038
关键词：超抗原；活化诱导细胞死亡；DNA片段化
免疫学杂志010105 摘 要：目的 为了探讨超抗原活化诱导CD4+T淋巴细胞凋亡分子机理及其信号传导途径，有必要建立超抗原活化诱导CD4+T细胞凋亡模型。方法 采用电镜观察细胞凋亡的形态学特征，借助流式细胞仪PI染色观察细胞凋亡的光散射特征及亚二倍体核型峰特征，琼脂糖凝胶电泳分析细胞凋亡的DNA片段化图谱，最后采用改进的二苯胺(DPA)法定量分析细胞凋亡DNA片段化百分率。结果 4μg/mLSEB刺激静息的SEB应答CD4+T细胞12h后，电镜下观察呈现典型的凋亡形态学特征；流式细胞仪PI染色分析表明，在二倍体峰的左侧出现亚二倍体核型峰典型凋亡特征，在光散射图谱上呈现低于正常细胞的前向散射和高于正常细胞的侧向散射；琼脂糖凝胶电泳分析呈现典型的凋亡DNA梯状图谱；DNA片段化百分率分析表明，超抗原SEB活化诱导CD4+T细胞凋亡率的升高具有时间依赖性，添加Fas-Ig融合蛋白能够特异性抑制凋亡DNA片段化百分率的升高。结论 超抗原SEB活化诱导CD4+T细胞凋亡模型的建立，为进一步深入探讨超抗原活化诱导T细胞死亡的分子机理和信号通路奠定了坚实基础。
, 百拇医药
分类号：R371-33；Q781 文献标识码：A
文章编号：1000-8861(2001)01-0017-05
Establishment of an apoptotic model on activation-induced CD4+T lymphocytes apoptosis mediated by superantigen SEB
XIONG Shi-qin(Department of Immunology，the Third Military Medical University，Chongqing 400038，China)
ZHU Xi-hua(Department of Immunology，the Third Military Medical University，Chongqing 400038，China)
, 百拇医药
HUANG Yun-hui(Department of Immunology，the Third Military Medical University，Chongqing 400038，China)
Abstract：Objective It is essential to establish the apoptotic model for exploration of the molecular mechanism and signaling pathways in activation-induced CD4+T lymphocytes apoptosis mediated by superantigen SEB.Methods The model of activation-induced CD4+T lymphocytes apoptosis by SEB was established according to electron micrograph of the apoptotic characteristics，DNA fluorescence flow cytometric profiles of PI-stained apoptotic CD4+T cells，the appearance of the nucleosomal DNA ladder in agarose gels，and the quantitative analysis of DNA fragmentation percent by improved DPA methods.Results There were typical morphological changes of apoptosis after SEB-responsive CD4+T lymphocytes stimulated with 4 μg/mL SEB for 12 hours.In addition，there was an apperance of apoptotic subdiploid peak，a higher side scatter and a lower forward scatter of typical apoptotic characteristics for SEB-induced apoptotic CD4+T lymphocytes than normal control.Moreover，analysis of the agarose gel eletrophoresis revealed that there was an apperance of typically apoptotic DNA ladder for SEB-induced apoptotic CD4+T lymphocytes.Finally，the kinetic increment of apoptosis was related to the time of stimulatation by SEB，and the increment of DNA fragmentation percent could be completely prevented by Fas-Ig fusion protein.Conclusion Our results strongly suggest that the apoptotic model might be a useful tool to explore the molecular mechansim and signaling transduction pathways in activation-induced CD4+T lymphocytes apoptosis mediated by superantigen SEB.
, 百拇医药
Keywords：superantigen；activation-induced cell death；DNA fragmentation
作者简介：熊世勤(1971-)，四川广安市人，讲师，博士，主要从事分子免疫学研究。现在北京清华大学生命科学与生物工程研究院基因组研究所，100084。 Tel：(010)62773625；E-mail：sqxiong＠chinaren.com
参考文献：
[1]Green DR，Scott DW.Activation-induced apoptosis in lymphocytes[J].Curr Opin Immunol，1994，6(3)：476-487.
[2]Spernt J，Webb SR.Intrathymic and extrathymic clonal deletion of T cells[J].Curr Opin Immunol，1995，7(2)：196-205.
, 百拇医药
[3]Mogil R L，Radvanyi L，Gonzalez-Quintial R，et al.Fas(CD95) participate in peripheral T cell deletion and associated apoptosis in vivo[J].Int Immunol，1995，7(9)：1 451-1 458.
[4]Lenardo MJ，Boehme L，Chen B，et al.Autocrine feedback death and the regulation of mature T lymphocytes antigen responses[J].Int Rev Immunol，1995，13(2)：115-134.
[5]Boise LA，Minn P，Noel C，et al.CD28 co-stimulation can promote T cell survival by enhancing the expression of Bcl-XL[J].Immunity，1995，3(1)：87-88.
, 百拇医药
[6]Bachmann MF，Ohashi PS.The role of T-cell receptor dimerization in T-cell activation[J].Immunol Today，1999，20(2)：576-586.
[7]Lavoie PM，Thibodeau J，Erard F，et al.Understand the mechanism of action of bacterial superantigens from a decade of research[J].Immunol Rev，1999，168：257-269
[8]Alderson MR，Tough TW，Davis-Smiss T，et al.Fas Ligand Mediates activation-induced cell death in human T lymphocytes[J].J Exp Med，1995，181(1)：71-77.
, 百拇医药
[9]Nicoletti I，Migliorati G，Pagliacci MC，et al.A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry[J].J Immunol Methods，1991，139(2)：271-279.
[10]熊世勤，朱锡华，黄云辉.一种快速分离细胞凋亡片段化DNA的简单方法——NP-40法[J].免疫学杂志，2000，16(3)：228-231.
[11]熊世勤.超抗原SEB激活诱导CD4+T淋巴细胞凋亡信号机制的研究[D].第三军医大学博士学位论文，2000.18-29.
收稿日期：2000年9月5日
出版日期：2001年1月15日, 百拇医药
单位：第三军医大学免疫学教研室，全军免疫学研究所，重庆 400038
关键词：超抗原；活化诱导细胞死亡；DNA片段化
免疫学杂志010105 摘 要：目的 为了探讨超抗原活化诱导CD4+T淋巴细胞凋亡分子机理及其信号传导途径，有必要建立超抗原活化诱导CD4+T细胞凋亡模型。方法 采用电镜观察细胞凋亡的形态学特征，借助流式细胞仪PI染色观察细胞凋亡的光散射特征及亚二倍体核型峰特征，琼脂糖凝胶电泳分析细胞凋亡的DNA片段化图谱，最后采用改进的二苯胺(DPA)法定量分析细胞凋亡DNA片段化百分率。结果 4μg/mLSEB刺激静息的SEB应答CD4+T细胞12h后，电镜下观察呈现典型的凋亡形态学特征；流式细胞仪PI染色分析表明，在二倍体峰的左侧出现亚二倍体核型峰典型凋亡特征，在光散射图谱上呈现低于正常细胞的前向散射和高于正常细胞的侧向散射；琼脂糖凝胶电泳分析呈现典型的凋亡DNA梯状图谱；DNA片段化百分率分析表明，超抗原SEB活化诱导CD4+T细胞凋亡率的升高具有时间依赖性，添加Fas-Ig融合蛋白能够特异性抑制凋亡DNA片段化百分率的升高。结论 超抗原SEB活化诱导CD4+T细胞凋亡模型的建立，为进一步深入探讨超抗原活化诱导T细胞死亡的分子机理和信号通路奠定了坚实基础。
, 百拇医药
分类号：R371-33；Q781 文献标识码：A
文章编号：1000-8861(2001)01-0017-05
Establishment of an apoptotic model on activation-induced CD4+T lymphocytes apoptosis mediated by superantigen SEB
XIONG Shi-qin(Department of Immunology，the Third Military Medical University，Chongqing 400038，China)
ZHU Xi-hua(Department of Immunology，the Third Military Medical University，Chongqing 400038，China)
, 百拇医药
HUANG Yun-hui(Department of Immunology，the Third Military Medical University，Chongqing 400038，China)
Abstract：Objective It is essential to establish the apoptotic model for exploration of the molecular mechanism and signaling pathways in activation-induced CD4+T lymphocytes apoptosis mediated by superantigen SEB.Methods The model of activation-induced CD4+T lymphocytes apoptosis by SEB was established according to electron micrograph of the apoptotic characteristics，DNA fluorescence flow cytometric profiles of PI-stained apoptotic CD4+T cells，the appearance of the nucleosomal DNA ladder in agarose gels，and the quantitative analysis of DNA fragmentation percent by improved DPA methods.Results There were typical morphological changes of apoptosis after SEB-responsive CD4+T lymphocytes stimulated with 4 μg/mL SEB for 12 hours.In addition，there was an apperance of apoptotic subdiploid peak，a higher side scatter and a lower forward scatter of typical apoptotic characteristics for SEB-induced apoptotic CD4+T lymphocytes than normal control.Moreover，analysis of the agarose gel eletrophoresis revealed that there was an apperance of typically apoptotic DNA ladder for SEB-induced apoptotic CD4+T lymphocytes.Finally，the kinetic increment of apoptosis was related to the time of stimulatation by SEB，and the increment of DNA fragmentation percent could be completely prevented by Fas-Ig fusion protein.Conclusion Our results strongly suggest that the apoptotic model might be a useful tool to explore the molecular mechansim and signaling transduction pathways in activation-induced CD4+T lymphocytes apoptosis mediated by superantigen SEB.
, 百拇医药
Keywords：superantigen；activation-induced cell death；DNA fragmentation
作者简介：熊世勤(1971-)，四川广安市人，讲师，博士，主要从事分子免疫学研究。现在北京清华大学生命科学与生物工程研究院基因组研究所，100084。 Tel：(010)62773625；E-mail：sqxiong＠chinaren.com
参考文献：
[1]Green DR，Scott DW.Activation-induced apoptosis in lymphocytes[J].Curr Opin Immunol，1994，6(3)：476-487.
[2]Spernt J，Webb SR.Intrathymic and extrathymic clonal deletion of T cells[J].Curr Opin Immunol，1995，7(2)：196-205.
, 百拇医药
[3]Mogil R L，Radvanyi L，Gonzalez-Quintial R，et al.Fas(CD95) participate in peripheral T cell deletion and associated apoptosis in vivo[J].Int Immunol，1995，7(9)：1 451-1 458.
[4]Lenardo MJ，Boehme L，Chen B，et al.Autocrine feedback death and the regulation of mature T lymphocytes antigen responses[J].Int Rev Immunol，1995，13(2)：115-134.
[5]Boise LA，Minn P，Noel C，et al.CD28 co-stimulation can promote T cell survival by enhancing the expression of Bcl-XL[J].Immunity，1995，3(1)：87-88.
, 百拇医药
[6]Bachmann MF，Ohashi PS.The role of T-cell receptor dimerization in T-cell activation[J].Immunol Today，1999，20(2)：576-586.
[7]Lavoie PM，Thibodeau J，Erard F，et al.Understand the mechanism of action of bacterial superantigens from a decade of research[J].Immunol Rev，1999，168：257-269
[8]Alderson MR，Tough TW，Davis-Smiss T，et al.Fas Ligand Mediates activation-induced cell death in human T lymphocytes[J].J Exp Med，1995，181(1)：71-77.
, 百拇医药
[9]Nicoletti I，Migliorati G，Pagliacci MC，et al.A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry[J].J Immunol Methods，1991，139(2)：271-279.
[10]熊世勤，朱锡华，黄云辉.一种快速分离细胞凋亡片段化DNA的简单方法——NP-40法[J].免疫学杂志，2000，16(3)：228-231.
[11]熊世勤.超抗原SEB激活诱导CD4+T淋巴细胞凋亡信号机制的研究[D].第三军医大学博士学位论文，2000.18-29.
收稿日期：2000年9月5日
出版日期：2001年1月15日, 百拇医药