【作者】 邓仲良；

【导师】 吴移谋；

【作者基本信息】 南华大学 ， 病原生物学， 2005， 硕士

【摘要】 目的 研究从解脲脲原体(Uu)提取的脂质相关膜蛋白(LAMPs)在体外诱导小鼠巨噬细胞表达诱导性一氧化氮合酶(iNOS)及产生一氧化氮(NO)的水平,研究该膜蛋白能否激活小鼠巨噬细胞中核因子kappa B(NF-κB)及诱导细胞发生凋亡的情况,以便了解Uu潜在的致病性,为进一步探讨Uu LAMPs诱导小鼠巨噬细胞表达iNOS及诱导巨噬细胞发生凋亡的分子机制提供实验依据。 方法 用从Uu提取的LAMPs刺激小鼠巨噬细胞,用Griess试剂测定经刺激后的小鼠巨噬细胞产生的NO水平,以RT-PCR和Western blot方法分析iNOS的表达水平,用细胞免疫组化、间接免疫荧光检测NF-κB的激活,用Western blot检测核提取物中NF-κB蛋白的表达,且利用RT-PCR和Western blot方法检测NF-κB的特异性抑制剂二硫代氨基甲酸吡咯烷(PDTC)和蛋白酶抑制剂放线菌酮(CHX)对iNOS的表达及对NF-κB激活的影响。用细胞凋亡检测试剂盒检测经Uu LAMPs处理的小鼠巨噬细胞的凋亡情况。 结果 Uu LAMPs能诱导小鼠巨噬细胞表达iNOS,且能以时间和剂量依赖方式刺激小鼠巨噬细胞产生NO,当LAMPs的浓度为3μg/mL时产生的NO量最多,为13.56±0.5μmol/L;但当LAMPs从3μg/mL增加到5μg/mL时,NO产生的量反而减少。另外当LAMPs刺激细胞4h后即可从培养基中检测到NO,而刺激32h后产生的NO量则达到高峰。在用该膜蛋白与NF-κB的抑制剂PDTC或蛋白酶抑制剂更多还原

【Abstract】 Objectives: The present study was undertaken to investigate the potential pathogenicity of ureaplasma urealyticum and the molecular mechanisms that is responsible for the induction of inducible nitric oxide synthase(iNOS) gene expression and cell apoptosis in mouse macrophages stimulated by lipid-associated membrane proteins (LAMPs) prepared from Ureaplasma urealyticum (Uu).LAMPs were utilized to study the expression of iNOS and the production of nitric oxide (NO); The activation of nuclear factor kappa B (NF-kB) and cell apoptosis in mouse macrophages stimulated by LAMPs were also detected.Methods: Mouse macrophages were stimulated with Uu LAMPs to assay NO with Griess Reagents,the expression of iNOS were detected by RT-PCR and western blot. The activation of NF-kB was examined in mouse macrophages treated with LAMPs by indirect immuno- fluorescence (IFA) , unmunocytochemistry and western blot.the effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kB and of cycloheximide (CHX), a protein synthase inhibitor, on the expression of iNOS and on the activation of NF-kB were also investivated in mouse macrophages treated with LAMPs by RT-PCR and western blotCell apoptosis was detected in mouse macrophages treated with LAMPs by Annexin-FITC Apoptosis Detection Kit.Results: Uu LAMPs stimulated mouse macrophages to express iNOS and thus produce NO in dose-and time-dependent manner. There was an optimal concentration of LAMPs at 3μg/mL in the induction of NO (13.56±0.54μmol/L), when the concentration of LAMPs added from 3μg/mL to 5μg/mL,whereas the production of NO decreased. The NO could be detected in the conditioned media after 4h by stimu-更多还原