RNA干扰技术抑制BAG-1在大鼠肺泡巨噬细胞表达及恢复GR抗炎效应的研究

INVESTIGATION OF REPRESSING BAG-1 EXPRESSION IN RAT ALVEOLAR MACROPHAGE WITH RNA INTERFERENCE TECHNIQUE AND RECOVERING ANTI-INFLAMMATORY EFFECTS OF GLUCOCORTICOID RECEPTOR

目的利用RNA干扰技术,建立脂多糖(LPS)和地塞米松(Dex)作用大鼠肺泡巨噬细胞BAG-1基因阻断模型,并阐明BAG-1表达对糖皮质激素受体(GR)抗炎活性的影响。方法构建两个针对BAG-1的RNAi质粒表达载体,分别命名为SiBAG-1α和SiBAG-1β,经测序确认后,转染大鼠肺泡巨噬细胞。细胞免疫组织化学法检测细胞核中BAG-1表达变化;电泳迁移率改变分析法(EMSA)检测GR活性变化。结果经测序证实:成功构建两个针对BAG-1基因的RNAi(RNA干扰)质粒表达载体SiBAG-1α和SiBAG-1β;转染质粒SiBAG-1α可明显降低胞核内BAG-1蛋白表达(P<0.05),而转染质粒SiBAG-1β对胞核内BAG-1蛋白表达无明显影响;质粒SiBAG-1α转染组细胞GR活性明显明显增强(P<0.05)。结论成功地构建并筛选出一个特异而高效地阻断BAG-1表达的质粒表达载体SiBAG-1α,抑制BAG-1表达可恢复GR抗炎活性,逆转GR活性下降所致的糖皮质激素抵抗。

Objective To establish a BAG-1 knockdown model of rat alveolar macrophages treated by lipopolysaceharide （LPS） and dexamethasone （Dex） with RNA interference technique, and elucidate the effects of BAG-1 expression on the anti-inflammatory activity of glueoeortieoid receptors. Methods Firstly two recombinant RNAi expression plasmids targeted on the BAG-1 gene （named SiBAG-1α and SiBAG-1β） were constructed, and confirmed by sequencing. The expression changes of BAG-1 protein in nuclei were evaluated with immunocytoehemistry after rat alveolar maerophages were transfeeted by RNAi plasmids; simultaneously GR activity was evaluated with eleetrophoretie mobility shift assay （EM- SA）. Results Plasmids SiBAG-1α and SiBAG-1β were successfully constructed and identified by sequencing. SiBAG-1α transfection specially inhibited BAG-1 protein expression in nuclei （P〈0. 05）, accompanied by the increase of GR activity （P〈0. 05）. SiBAG-113 transfeetion had no significant influence on BAG-1 protein expression and GR activity, compared to the normal control. Conclusion A recombinant RNAi expression plasmid SiBAG-lu which can specifically and efficiently knockdown BAG-1 gene is sueeessfully constructed. Repressing BAG-1 expression can bring the recovery of anti-inflammatory activity of GR, which can reverse glucocorticoid resistance induced by the decrease of GR activity.