【作者】 吴伟；

【导师】 李大主；

【作者基本信息】 华中科技大学 ， 心血管内科， 2006， 硕士

【摘要】 第一部分热休克蛋白60对小鼠树突状细胞成熟及免疫功能研究的体外研究目的探讨动脉粥样硬化中重要的炎性物质——热休克蛋白60(HSP60)体外对小鼠树突状细胞(mDC)功能的影响。方法从近交系C57BL/6J小鼠骨髓提取DC,体外培养成熟后分别与5μg/ml及10μg/ml mHSP60、OVA或PBS孵育24h,观察各组mDC的形态学改变;流式细胞仪(FCA)检测各组mDC的CD80及CD86表型表达;混合淋巴细胞反应(MLR)测定各组mDC刺激T淋巴细胞增值能力;ELISA法测定MLR的上清液中细胞因子IL-4、IL-12与IFN-γ浓度。结果OVA组与mHSP60组与PBS组比较,mDC的突起增加明显;CD80及CD86表型显著增加;T淋巴细胞刺激功能明显增强;MLR的上清液细胞因子IL-12、IFN-γ增加(P<0.01)而IL-4增加不明显(P>0.05),IFN-γ/IL-4比值升高。OVA组较HSP60各组效应更加明显,而高剂量HSP60组较低剂量HSP60组效应明显。结论mHSP60体外可以促进mDC免疫激活功能,且作用呈剂量依赖性。第二部分负载HSP60树突状细胞对ApoE-/-小鼠动脉粥样斑块影响目的探讨负载热休克蛋白60(HSP60)的树突状细胞(DC)接种ApoE-/-小鼠对主动脉粥样硬化斑块的影响。方法小鼠髓源性DC(mDC)体外培养,分别用PBS、OVA和HSP60处理至成熟;流式细胞仪(FCA)检测各组mDC表达CD80与CD86的水平以了解mDC成熟程度;同系ApoE-/-小鼠予以高脂饮食16周后形成粥样斑块,各组mDC用荧光物质CFSE标记后,分别于小鼠腹部经皮接种三次,48小时后取主动脉HE染色及荧光观察,测血清中IL-10、IFN-γ浓度。结果体外HSP60及OVA可促进mDC表达CD80与CD86水平,而PBS则无此效应。接种同系ApoE-/-小鼠后,HSP60-DC和OVA-DC组血清IFN-γ增高;而IL-10无明显差别;IFN-γ/IL-10比值增高。HSP60-DC促使主动脉粥样斑块炎性细胞浸润明显增加,斑块趋于不稳定;OVA-DC与PBS-DC则对斑块无显著效应。HSP60-DC迁移至主动脉粥样斑块的数目较OVA-DC与PBS-DC明显增加(P<0.01)结论HSP60负载的DC可以靶向迁移至主动脉粥样斑块,特异性刺激斑块的细胞免疫反应,加重炎性反应,诱导炎性细胞因子释放,引起免疫偏移。更多还原

【Abstract】 Part 1 Effect of heat shock protein 60 on murine dendritic cell maturation and immunologic function in vitroObjective To investigate the effect of heat shock protein 60 (HSP60), significant inflammatory factor in atherogenesis, on the maturation of murine dendritic cells (mDCs) in vitro. Methods Myeloid mDCs were obtained in vitro from C57BL/6J mouse marrow and incubated with OVA, two concertrations of mHSP60 or PBS 24 hours, observed the morphological changes; detected the variation of CD80 and CD86 phenotype with flow cytometric analysis (FCA); the stimulating T-cell capacity determined in allogenic mixed lymphocyte reaction (MLR); ELISA was used to analyze the level of cytokines, such as IL-4, IL-12 and IFN-γin the medium of MLR. Results Compared to control group, mDC showed more protrusions in OVA and mHSP60 incubated groups. The expression of CD86, CD80 on OVA-DCs and mHSP-DCs up-regulated markedly. The stimulating T-cell capacity of OVA-DCs and mHSP-DCs was higher. Higher levels of IFN-γ, IL-12 but no significant difference of IL-4 was displayed in supernatant of MLR and the ratio of IFN-γ/ IL-4 was higher. Above effects were evident on OVA-DCs and higher mHSP60 concentration. Conclusion mHSP60 promotes the maturation of mDC and the capacity of immune activity dose-dependently in vitro. Part 2 Target Effect of Heat Shock Protein 60-loaded Dendritic Cells on Progression of Atherosclerosis in Apolipoprotein E-/- MiceObjective To investigate whether murine dendritic cells (mDCs) loaded with heat shock protein 60(HSP60) could enhance atherosclerotic plaque in Apolipoprotein E-null mice. Methods Myeloid mDCs were extracted and incubated in vitro then incubated with PBS, OVA and HSP60 to maturation respectively and observed the morphological changes and detected the variation of CD80 and CD86 phenotype with flow cytometric analysis (FCA) in vitro to know the degree of mDCs maturation. Homogenous mice were fed high-cholesterol diet 16 weeks to form plaque. ApoE-/- mice were treated with mDCs marked by fluorescent CFSE subcutaneously for three times at a one–week interval. 48 hours after the last treated, aortas were harvested for Haematoxylin-Eosin stained and anti-CD4+T cell immunostained. Serum cytokines (IL-10, IFN-γ) were analyzed by ELISA assay. Results Compared to PBS-DC group, HSP60-DC and OVA-DC group expressed higher levels of CD80 and CD86. After treatment of mice with DCs, serum IFN-γlevel was increased in HSP60-DC and OVA-DC group (P<0.01), while IL-10 no significance (P>0.05), and IFN-γ/IL-10 ratio also rose (P<0.01). HSP60-DC made inflammatory cells infiltration in atherosclerotic plaques greatly, and plaques tended to vulnerability. Then OVA-DC had no significance to plaque (P>0.05). And HSP60-DC migrated to atherosclerotic plaques more greatly than OVA-DC and PBS-DC (P<0.01). Conclusion DC loaded HSP60 enhances inflammatory reaction of atherosclerotic plaque and induces cytokines release, leads to immune deviation.更多还原