【作者】 杨克平；

【导师】 李大主；

【作者基本信息】 华中科技大学 ， 心血管内科， 2007， 硕士

【摘要】 ApoE-/-小鼠CD4+CD25+ T细胞分离及功能检测目的探讨ApoE-/-小鼠CD4+CD25+T细胞的比例和功能及其与动脉粥样硬化的关系。方法从apoE-/-小鼠外周血流式细胞仪(FCA)检测CD4+CD25+T细胞的比例并分分选之;混合淋巴细胞反应(MLR)研究CD4+CD25+T细胞的免疫抑制功能;ELISA法测定上清液中细胞因子IL-10、TGF-β浓度;观察ApoE-/-小鼠动脉粥样斑块的形成状况。结果与正常小鼠相比,ApoE-/-小鼠外周血中CD4+CD25+T细胞数量无差异,但抑制naive T细胞增殖能力弱,分泌更少IL-10、TGF-β;ApoE-/-小鼠斑块面积较大。结论ApoE-/-小鼠CD4+CD25+T细胞抑制功能弱,可能因此免疫失稳而致动脉粥样硬化。HSP60特异性调节性T细胞的诱导及其对小鼠动脉粥样硬化影响目的探讨抗原特异性CD4+CD25+T细胞的体外诱导及其对动脉粥样硬化斑块形成的影响。方法从apoE-/-小鼠分离骨髓单个核细胞,经雷帕霉素(RPM)处理培养出未成熟树突状细胞(DC);体外诱导HSP60特异性调节性T细胞分化;流式细胞仪(FCA)检测及分选CD4+CD25+T细胞;混合淋巴细胞反应(MLR)研究CD4+CD25+T细胞的特异性抑制效应;ELISA法测定上清液中细胞因子IL-10、TGF-β和IFN-γ浓度。过继转移CD4+CD25+T细胞后,观察小鼠动脉粥样斑块的形成状况。结果雷帕霉素处理的DC共刺激分子CD80和CD86表达明显减少,形态学表现为未成熟树突状细胞;未成熟树突状细胞比成熟树突状细胞能诱导更多CD4+CD25+T细胞;培养体系中细胞因子IL-10、TGF-β水平明显升高;CD4+CD25+T细胞能抑制效应性T细胞的增殖及IFN-γ的分泌。过继HSP60特异性CD4+CD25+T细胞组小鼠斑块面积较小。结论未成熟树突状细胞可诱导出抑制功能强大的HSP60特异性CD4+CD25+T细胞,后者在体内能能明显抑制斑块的形成。更多还原

【Abstract】 Isolation and functional characterization of CD4+ CD25+ Treg cells from apoE-/- miceObjective To explore the ratio and function of CD4+CD25+T cell in apoE-/- mice and its relation with atherosclerosis. Methods FCA were used to detect and select CD4+CD25+T cell from peripheral blood of apoE-/- mice. The CD4+CD25+T cells’inhibition were investigated through mixed lymphocyte reaction. Cytokines in the supernatant were analysed by ELISA assay. The state of atheromatous plaque were observed. Results Compare to normal mice, CD4+CD25+T cell in apoE-/- mice had similar ratios and activited weaker inhibition and secreted less IL-10 and TGF-β. The plaques of the apoE-/- mice were larger than normal mice. Conclusion CD4+CD25+ T cells in apoE-/- mice lose its suppressive characterization and thus break immunological homeostasis and then cause atherosclerosis. Generation of HSP60-Specific Regulatory T cell and Effect on Atherosclerosis in miceObjective To explore induction of antigen-specific-CD4+CD25+T cell in vitro and its effect on the formation of plaque. Methods Bone marrow monouclear cells were extracted from apoE-/- mouse and cultured into immature dendritic cells by RPM and then were used to induce heat shock protein 60-specific-CD4+CD25+T cells in vitro. FCA were used to detect and select CD4+CD25+T cell. The CD4+CD25+T cells’specific inhibition were investigated through mixed lymphocyte reaction. Cytokines in the supernatant were analysed by ELISA assay. After infusing the CD4+CD25+T cells into homogenous mice, we observed the state of the plaques. Results The expression of co-stimulating factor CD80, CD86 on RPM-treated-dendritic cells was down-regulated. Immature dendritic cells could be used to induce more antigen-specific-CD4+CD25+T cells than mature ones and IL-10 and TGF-βlevels in the culture medium were higher. These CD4+CD25+T cells could suppress the proliferation and the IFN-γproduction of effector T cells in vitro notablly. The plaques of the mice which were infused CD4+CD25+T cells were smaller than those uninfused. Conclusion Immature dendritic cells could be used to induce suppressive antigen-specific-CD4+CD25+T cells which could affect the formation of plaques.更多还原