下调PALM3表达抑制脂多糖诱导的巨噬细胞炎性反应  

Downregulation of Paralemmin-3 Expression Inhibits the Inflammatory Reaction Induced by LPS in Alveolar Macrophages

作　　者：陈旭昕[1] 宋立成 孟激光[1] 韩志海[1] Chen Xuxin;Song Licheng;Meng Jiguang(Department of Respiratory Medicine,Navy General Hospital of PLA ,Beifing 100048,China)

机构地区：[1]中国人民解放军海军总医院呼吸内科,北京100048

出　　处：《医学研究杂志》2018年第12期22-26,共5页Journal of Medical Research

基　　金：国家自然科学基金资助项目(81300050);北京市自然科学基金资助项目(7182163);中国人民解放军海军总医院创新培养基金资助项目(CXPY201417)

摘　　要：目的探讨抑制paralemmin-3(PALM3)表达对脂多糖(LPS)诱导的大鼠肺泡巨噬细胞系(NR8383)炎性反应的影响。方法将NR8383细胞并接种至6孔板中,待融合度达70%,加入0.5μg/ml LPS刺激12h,用免疫荧光检测细胞PALM3表达及亚定位;用0. 5μg/ml LPS刺激NR8383细胞并在0、3、6、12、24和48h收集细胞提取总RNA,用RT-PCR法检测PALM3 mRNA;同前接种细胞至24孔板,待融合度达70%,将PALM3小干扰核糖核苷酸(siRNA)转染NR8383细胞(PALM3 siRNA转染组),同时设立正常NR8383细胞组和对照转染组(转染control siRNA),转染48h后,提细胞总蛋白用Western blot法检测PALM3蛋白水平;3组细胞给予LPS刺激12h,收集培养上清和核蛋白,用ELISA法检测培养上清中IL-8、IL-6的水平及核转录因子(NF-κB)转录活性。结果 PALM3分子在NR8383细胞中有表达,LPS可诱导PALM3表达;PALM3 siRNA转染后成功下调PALM3蛋白表达,予LPS刺激后,与正常细胞及对照转染组比较,PALM3 siRNA转染组培养上清中白介素-8(IL-8)和IL-6含量减少,PALM3 siRNA转染组细胞中NF-KB转录活性亦被抑制。结论 NR8383细胞中有PALM3表达,LPS可诱导NR8383细胞中PALM3的蛋白表达,下调PALM3表达可抑制LPS诱导的炎性反应。Objective To investigate the effect of downregulation of paralemmin -3 (PALM3)expression on the LPS-induced inflammatory reaction in rat alveolar macrophages (NR8383 cells).Methods NR8383 cells were conventionally cultured and seeded in 6-well plates.After the cell growth reached 70% confluence,NR8383 cells were treated with 0.5μg/ml for 12h.The expression and localization of PALM3 in NR8383 cells was determined by confocal immunofluorescence.And,the mRNA level of PALM3 were detected by RT -PCR at 0,3,6,12,24 and 48h post LPS -stimulation.Another NR8383 cells were seeded in 24-well plates.When the cell growth reached 70%confluence,the cells were transfected by PALM3 siRNA.Meanwhile,an empty vector was used as a negative control group and non-transfection NR8383 cells was used as a normal control group.At 48h after transfection,the protein expression of PALM3 in NR8383 cells was determined by Western blot analysis.LPS was administered into the cell culture medium at 48h post-transfection. The culture supernatant and nucleoprotein were collected at 12h after LPS-stimulation.The concentrations of interleukin -8 (IL-8 ) and IL-6 and the activity of nuclear transcrition factor-κB (NF -κB)were measured by ELISA.Results PALM3 was expressed in NR8383 cells and LPS could upregulate the PALM3 expression in a time-dependent manner.PALM3 siRNA could suppress the expression of PALM3 in NR8383 cells effectively.Downregulation of PALM3 expression could significantly inhibit the production of IL-8 and IL -6 and the activation of NF-κB induced by LPS in NR8383 cells.Conclusion PALM3 are expressed in rat alveolar macrophages (NR8383 cells)and LPS can cause PALM3-induced expression.Downregulation of PALM3 expression can ameliorate the inflammatory reactions to LPS in NR8383 cells.

关 键 词：Paralemmin-3 脂多糖 巨噬细胞 炎症 

分 类 号：R563[医药卫生—呼吸系统]