RNA干扰技术对人U937巨噬细胞株糖皮质激素受体表达及功能的抑制效应  

Repressive effects of RNA interference technique on expression and function of glucocorticoid receptor in human macrophage cell line U937

作　　者：张芳[1] 钱桂生[1] 陈维中[1] 苏踊跃[2] 

机构地区：[1]第三军医大学新桥医院全军呼吸内科研究所,全军呼吸病研究重点实验室,重庆400037 [2]第三军医大学西南医院全军烧伤研究所,创伤,烧伤与复合伤国家重点实验室,重庆400038

出　　处：《第三军医大学学报》2005年第10期951-953,共3页Journal of Third Military Medical University

摘　　要：目的　利用载体介导的RNA干扰技术,建立糖皮质激素受体(GR)基因阻断的人U93 7巨噬细胞株模型。方法　构建两个针对GR的RNAi重组表达载体,分别命名为pSilencer 3 1 GR1和pSilencer 3 1 GR2 ,经测序确认后,转染人U93 7巨噬细胞株。RT PCR、Westernblot分别检测糖皮质激素受体mRNA水平和蛋白水平表达变化;相对荧光素酶活性法检测地塞米松作用后GR转录激活功能的变化。结果　经测序证实:成功构建两个针对GR的RNAi表达载体(pSilencer3 1 GR1和pSilencer 3 .1 GR2 ) ;转染pSilencer 3 .1 GR2 可明显降低细胞内GRmRNA的丰度及GR蛋白表达,细胞内GR转录激活功能明显降低;转染pSilencer 3 .1 GR1与对照组相比无显著变化。结论　成功地构建并筛选出一个高效且特异阻断GR表达及功能的RNAi质粒表达载体,为进一步研究糖皮质激素受体的功能提供新方法。Objective To establish a glucocorticoid receptor knockdown model of human macrophage cell line U937 with RNA interference technique. Methods Two RNAi recombinant plasmids (named pSilencer 3.1-GR 1 and pSilencer 3.1-GR 2) targeting to GR gene were constructed. After RNAi recombinant plasmids were transfected into human macrophage cell line U937, the expressions of GR mRNA and GR protein were evaluated with RT-PCR and Western blotting respectively. The transcriptional activation function of GR was evaluated through the detection of relative luciferase activity after dexamethasone treatment. Results Two RNAi recombinant expression plamids were constructed and identified by sequencing. pSilencer 3.1-GR 2 transfection could inhibit not only GR mRNA and protein expressions, but also transcriptional activation function of GR specially; pSilencer 3.1-GR 1 transfection had no significant changes as compared to normal control. Conclusion A glucocorticoid receptor knockdown model has been established successfully, which offers a new method for the further research of GR biological functions.

关 键 词：糖皮质激素受体 人巨噬细胞株U937 RNA干扰 

分 类 号：R392.12[医药卫生—免疫学] R394-33[医药卫生—基础医学]