目的 研究CDla和CD83分子是否特异性地表达在乳腺癌患者外周血中培养的树突状细胞上。方法 采用密度梯度离心的方法，分离32例乳腺癌患者外周血中的单个核细胞，在6孔培养板上(10^6／ml，2ml／孔)，用含10％热灭火的小牛血清和GM-CSF、I...目的 研究CDla和CD83分子是否特异性地表达在乳腺癌患者外周血中培养的树突状细胞上。方法 采用密度梯度离心的方法，分离32例乳腺癌患者外周血中的单个核细胞，在6孔培养板上(10^6／ml，2ml／孔)，用含10％热灭火的小牛血清和GM-CSF、IL-4、TNF-α细胞因子的RPMI 1640培养基培养。(贴壁)2小时后，轻轻吸去非贴壁细胞并加入新鲜培养基。用标有荧光素的单抗标记培养细胞，在流式细胞仪上分析培养细胞，本研究中所用的单抗有异硫氰酸荧光素(FITC)标记的CDla、CD3、CDl9、CD40、CD80和藻红朊(PE)标记的CD3、CDl9、CD83、CD86、MHC Ⅱ。在激光共聚焦显微镜下拍摄标记有荧光素的细胞。结果 该细胞表达特征性分子CDla、CD40、CD80、CD83、CD86，CDla、CD83在CD3+T、CDl9+B淋巴细胞上也表达，在激活的淋巴细胞上表达水平高于未激活的淋巴细胞，而且B淋巴细胞上的表达水平高于T淋巴细胞。结论 CDla和CD83分子并非DC特有的标记，目前鉴定DC仍然要靠细胞形态、细胞表达CDla、CD83以及共刺激分子等综合因素来确定DC。 Objective To study the specificity of molecules CD1 a and CD83 expression on the cultured dendritic cells in the peripheral blood of breast cancer patients.Methods Mononuclear cells of peripheral blood were isolated by Ficoll Hypaque centrifugation from 32 breast cancer patients and plated on 6-well culture plates( 106/ml, 2 ml/well) in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum, GM-CSF, IL-4, and/or TNF-et. Two h later, nonadherent cells were gently removed and fresh medium was added. Cultured cells were analyzed by flow cytometry with fluorescence labeled monoclonal antibodies (mAbs). The monoelonal antibodies used in this study included fluoreseein isothiocyanate (FITC)-eonjugated mAbs specific for CDla, CD3, CD19, CD40, and CD80; phycoerythrin (PE)-conjugated mAbs specific for CD3, CD19, CD83, CD86, and MHC-I1. Cultured and fluorescence stained cells were taken pictures by confocal scanning microscope respectively.Results Cells displayed a characteristic phenotype such as CDla+ , CD40+ , CD80+ , CD86+ and CD83+. CDla and CD83 molecules could also be expressed on the surface of CD3+ T lymphocytes and CD19+ B lymphocytes. The expression levels of CDla and CD83 in activated lymphocytes were higher than those in the unactivated lymphocytes, and higher in B lymphoeytes than in T lymphocytes.Conclusion The molecules of CD1 a and CD83 are not specific phenotypes for dendritic cells. We now still need to apply both cell morphology and molecules such as CDla, CD40, CD80, CD83 and CD86 to identify dendritic cells.展开更多
文摘目的 研究CDla和CD83分子是否特异性地表达在乳腺癌患者外周血中培养的树突状细胞上。方法 采用密度梯度离心的方法，分离32例乳腺癌患者外周血中的单个核细胞，在6孔培养板上(10^6／ml，2ml／孔)，用含10％热灭火的小牛血清和GM-CSF、IL-4、TNF-α细胞因子的RPMI 1640培养基培养。(贴壁)2小时后，轻轻吸去非贴壁细胞并加入新鲜培养基。用标有荧光素的单抗标记培养细胞，在流式细胞仪上分析培养细胞，本研究中所用的单抗有异硫氰酸荧光素(FITC)标记的CDla、CD3、CDl9、CD40、CD80和藻红朊(PE)标记的CD3、CDl9、CD83、CD86、MHC Ⅱ。在激光共聚焦显微镜下拍摄标记有荧光素的细胞。结果 该细胞表达特征性分子CDla、CD40、CD80、CD83、CD86，CDla、CD83在CD3+T、CDl9+B淋巴细胞上也表达，在激活的淋巴细胞上表达水平高于未激活的淋巴细胞，而且B淋巴细胞上的表达水平高于T淋巴细胞。结论 CDla和CD83分子并非DC特有的标记，目前鉴定DC仍然要靠细胞形态、细胞表达CDla、CD83以及共刺激分子等综合因素来确定DC。 Objective To study the specificity of molecules CD1 a and CD83 expression on the cultured dendritic cells in the peripheral blood of breast cancer patients.Methods Mononuclear cells of peripheral blood were isolated by Ficoll Hypaque centrifugation from 32 breast cancer patients and plated on 6-well culture plates( 106/ml, 2 ml/well) in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum, GM-CSF, IL-4, and/or TNF-et. Two h later, nonadherent cells were gently removed and fresh medium was added. Cultured cells were analyzed by flow cytometry with fluorescence labeled monoclonal antibodies (mAbs). The monoelonal antibodies used in this study included fluoreseein isothiocyanate (FITC)-eonjugated mAbs specific for CDla, CD3, CD19, CD40, and CD80; phycoerythrin (PE)-conjugated mAbs specific for CD3, CD19, CD83, CD86, and MHC-I1. Cultured and fluorescence stained cells were taken pictures by confocal scanning microscope respectively.Results Cells displayed a characteristic phenotype such as CDla+ , CD40+ , CD80+ , CD86+ and CD83+. CDla and CD83 molecules could also be expressed on the surface of CD3+ T lymphocytes and CD19+ B lymphocytes. The expression levels of CDla and CD83 in activated lymphocytes were higher than those in the unactivated lymphocytes, and higher in B lymphoeytes than in T lymphocytes.Conclusion The molecules of CD1 a and CD83 are not specific phenotypes for dendritic cells. We now still need to apply both cell morphology and molecules such as CDla, CD40, CD80, CD83 and CD86 to identify dendritic cells.
