肺炎支原体lamps经pi3knrf2诱导人单核细胞表达ho-1word论文.docxVIP

肺炎支原体 LAMPs 经 PI3K/Nrf2 诱导人单核细胞表达 HO-1研究生：刘艳导师：朱翠明教授中文摘要目的探讨肺炎支原体（ Mycoplasma pneumoniae ， Mp ）脂质相关膜蛋白 （lipid-associated membrane proteins，LAMPs）对人单核细胞表达血红素氧合 酶-1（heme oxygenase-1，HO-1）的影响，并探讨可能的调控机制。方法体外培养 THP-1 细胞，用不同浓度的 LAMPs 作用后，分别采用 realtime-PCR 和 Western blot 检测 HO-1 mRNA 和蛋白的表达。同时采用不同浓度的放线菌素 D（ActD）和放线菌酮（CHX）预处理细胞，观察其对 HO-1 表达的影响，以证 实 HO-1 的表达是否通过转录和翻译水平；提取 LAMPs 作用前后的核蛋白，凝 胶迁移率实验检测 Nrf2 的核转位、Western blot 检测 Akt 的磷酸化情况；同时采 用 PI3K 抑制剂 LY294002 处理细胞，观察其对 LAMPs 诱导 Nrf2 核转位及 HO-1 表达的影响；最后采用 siRNA 干扰 Nrf2 表达，以证实 Nrf2 是否参与调控 HO-1 表达。结果(1)0～5μg/mL LAMPs 能以剂量依赖性方式诱导 THP-1 表达 HO-1 mRNA 和蛋 白；(2)5μg/mL LAMPs 能诱导 THP-1 细胞 Akt 磷酸化，并能增强其 DNA 结合活性； PI3K 抑制剂 LY294002 处理后，可明显抑制 LAMPs 诱导的 HO-1 表达和 Nrf2 核转位；(3)干扰 Nrf2 表达后，可明显下调 LAMPs 诱导 THP-1 细胞表达 HO-1；1结论LAMPs 可诱导 THP-1 细胞表达 HO-1，其机制可能与 PI3K/Nrf2 通路有关。关键词肺炎支原体；脂质相关膜蛋白；血红素氧合酶-1；核因子相关因子-2;本课题受湖南省自然科学基金项目（NO：14JJ2090）资助。2英文摘要Mycoplasma pneumonia-derived lipid-associated membrane proteins induce Heme oxygenase-1 expression via PI3K/Nrf2 in THP-1 cellsObjectiveABSTRACTThe aim of this study was to investigate the mechanisms of Mycoplasma pneumonia-derived lipid-associated membrane proteins (LAMPs) inducing the expression of heme oxygenase-1 in human mononuclear cell line THP-1 cells.MethodsTHP-1 cells were cultured in vitro, and treated by different concentration of LAMPs, the mRNA and protein level of HO-1 were detected by realtime-PCR and western blot, respectively. In the meanwhile, actinomycin D and cycloheximide were administered to pretreat THP-1 cell, subsequently challenged by 5.0μg/mL LAMPs for 16h, and the expression of HO-1 was detected by western blot. To determine the phosphorylation of Akt and the DNA-binding activity of Nrf2, western blot and electrophoretic mobility shift assay were used to detect the nucleoprotein and cytoplasm protein extracted from the THP-1 cell administered by LAMPs. The PI3K inhibitor LY294002 was also administered to investigate the effects of LAMPs on the activity of Nrf2. At last, siRNA was