BACKGROUND AND OBJECTIVES:It is well documented that the decrease of glucocorticoid receptor (GR) can lead to Yin-yang Deficiency Syndrome in the Traditional Chinese Medicine theory. Youguiyin a famous and traditional Chinese medicine is often used to treat Yang Deficiency Syndrome, but its mechanism of action and target remains unknown. We aimed to establish one cell model whose the GR gene had been decreased and observe the effect of the traditional Chinese medicine Youguiyin on GR gene expression at mRNA level. MATERIALS AND METHODS:Established recombinant plasmids of GR gene by combination use RNAi and the Cre-LoxP system, stably transfected the recombinant plasmids and Cre-ERT2 plasmid into the murine macrophage RAW264.7 cells and selected with G418 and hygromycin B respectively, then used 4-TH to induce Cre-ERT2 plasmid to express. RT-PCR and Western blot methods to validate the change of GR gene at mRNA and protein level were employed. Feed orally SD male rats with Youguiyin and got their blood serum, used these blood serum to culture RAW264.7-Cre-GR (-,F) cells, detected the change of GR gene at mRNA level by RT-PCR. RESULTS:We successfully constructed two recombinant plasmids of GR gene which can make GR gene's expression to decrease significantly in RAW264.7 cells. The blood serum which contained Youguiyin can enhance the expression of GR mRNA. CONCLUSIONS:Combination use RNAi and the Cre-LoxP system can decrease GR gene's expression in the murine macrophage RAW264.7 cells. Youguiyin can enhance the expression of GR mRNA.

译文

背景与目的:已有大量文献报道,糖皮质激素受体(GR)的降低可导致中医理论的阴阳虚证。右归饮是一种著名的传统中药,常被用于治疗阳虚证,但其作用机理和靶向作用尚不清楚。我们的目的是建立一个GR基因减少的细胞模型,并观察中药油桂银在mRNA水平上对GR基因表达的影响。
材料与方法:利用RNAi和Cre-LoxP系统联合建立GR基因重组质粒,将重组质粒和Cre-ERT2质粒稳定转染到鼠巨噬细胞RAW264.7细胞中,分别用G418和潮霉素B筛选,然后使用4-TH诱导Cre-ERT2质粒表达。采用RT-PCR和Western blot方法验证GR基因在mRNA和蛋白质水平的变化。用尤桂银口服SD雄性大鼠,取血清,用这些血清培养RAW264.7-Cre-GR(-,F)细胞,通过RT-PCR检测GR基因在mRNA水平的变化。
结果:成功构建了两个GR基因重组质粒,可以使GR基因在RAW264.7细胞中的表达显着降低。含有幽归饮的血清可以增强GR mRNA的表达。
结论:RNAi和Cre-LoxP系统联合使用可降低GR基因在小鼠巨噬细胞RAW264.7细胞中的表达。右归饮可以增强GR mRNA的表达。

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