摘要
目的探讨X盒结合蛋白1(X-box binding protein1,XBP1)在低浓度皮质酮对小鼠腹腔巨噬细胞免疫功能调控中的作用。方法分离培养成年健康雄性C57BL/6小鼠腹腔巨噬细胞,应用XBP1-RNAi慢病毒或阴性对照慢病毒感染小鼠腹腔巨噬细胞,采用荧光显微镜观察感染效率,感染3、5d后收集巨噬细胞提取总RNA,应用RT-PCR检测小鼠腹腔巨噬细胞XBP1mRNA表达水平和干扰效率。XBP1-RNAi慢病毒或阴性对照慢病毒感染离体培养的小鼠腹腔巨噬细胞3d后,再应用低浓度皮质酮(50ng/ml)处理小鼠腹腔巨噬细胞1h,分别应用ELISA法及激光共聚焦显微镜检测小鼠腹腔巨噬细胞培养上清中TNF-α生成及其吞噬功能变化。结果当复感染指数(multiplicity of infection,MOI)值≥6时,XBP1-RNAi慢病毒能有效地感染小鼠腹腔巨噬细胞,感染效率>75%。应用RT-PCR检测发现XBP1-RNAi能有效地抑制小鼠腹腔巨噬细胞XBP1基因表达。通过选择性地沉默XBP1基因表达,低浓度皮质酮(50ng/ml)对巨噬细胞吞噬功能增强的作用受到抑制,巨噬细胞的吞噬率和吞噬指数均显著下降(P<0.01),而且对TNF-α分泌增强的作用也受到抑制,显著低于阴性对照慢病毒组(P<0.01)。结论采用RNA干扰选择性地沉默XBP1,能有效地抑制低浓度皮质酮对巨噬细胞吞噬和TNF-α生成增强的作用。
Objective To explore the role of X-box binding protein 1 （XBP1） in immune function regulation of macrophage by low conceatration of corticosterone. Methods Peritoneal macrophages were isolated from adult male C57BL/6 mice, and then infected with XBP1-RNAi lentivirus or negative control lentivirus. The infection efficiency was observed by fluorescence microscopy. Mice peritoneal macrophages were collected in 3 and 5 d after infection and total RNA were extracted. RT-PCR analysis was performed to detect the effect of RNA interference. Furthermore, mice peritoneal macrophages were cultured in vitro and infected with XBP1-RNAi lentivirus or negative control lentivirus for 3 d, and then treated with low concentration of corticosterone （50 ng/ml） for 1 h. TNF-α concentration in the cell culture supernatant and phagocytosis of macrophages were determined by enzyme-linked immunosorbent assay （ELISA） and confocal microscopy, respectively. Results When the multiplicity of infection （MOI） was more than 6, the infection efficiency of XBP1-RNAi lentivirus was above 75% after infection 4 d. RT-PCR analysis confirmed that XBP1-RNAi lentivirus effectively inhibited the expression of XBP1 mRNA in mice peritoneal macrophages. Furthermore, after selectively silencing of the XBP1 gene, the enhancement of macrophage phagocytosis by low concentration of corticosterone was efficiently inhibited, phagocytic rate and phagocytic index of mice peritoneal macrophages were decreased markedly compared with that of negative control lentivirus group （P0.01）. Meanwhile, the enhancement of TNF-α production by low concentration of corticosterone was also depressed, the concentration of TNF-α in the cell culture supernatant was significantly declined in XBP-RNAi lentivirus group, compared with the negative control lentivirus group （P0.01）. Conclusion Selective silencing of XBP1 by RNA interference may effectively inhibit the enhancement of macrophage phagocytosis and TNF-α production by low concentration of corticosterone.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2009年第20期1964-1968,共5页
Journal of Third Military Medical University
基金
国家重点基础研究发展计划(973计划)(2005CB522602)
全军医学科研“十一五”计划科技攻关项目(06G080)~~
关键词
糖皮质激素
巨噬细胞
RNA干扰
XBP1
免疫功能
glucocorticoids
macrophage
RNA interference
X-box binding protein 1
immune function
