人K562细胞糖皮质激素受体基因阻断模型的建立

Construction of glucocorticoid receptor gene block model in human K562 cells

目的利用腺病毒载体介导的RNAi技术,建立糖皮质激素受体基因阻断的人K562细胞模型。方法将针对糖皮质激素受体发挥RNAi效应的合成寡核苷酸序列连入含有绿色荧光蛋白基因的腺病毒穿梭质粒pRNAT-H1·1/Adeno中,在含有pAdEasy-I病毒骨架的BJ5183大肠菌内进行同源重组;重组体通过脂质体介导转染293T细胞,并在293细胞内包装为具有感染能力的病毒颗粒,通过反复感染扩增病毒以达感染靶细胞的适当滴度,通过绿色荧光表达来监测腺病毒扩增;应用Western blot法检测人K562细胞感染病毒后糖皮质激素受体蛋白的表达。结果重组腺病毒穿梭质粒psiGR/Adeno和重组腺病毒pAdEasy-siGR的成功构建;转染腺病毒载体的293T细胞表达绿色荧光,随着时间逐渐增强并且出现明显的细胞病变效应,经过3轮扩增,病毒达到合适的滴度;受腺病毒感染的K562细胞内糖皮质激素受体蛋白表达明显降低。结论可以成功构建针对糖皮质激素受体基因的RNAi腺病毒载体,为进一步研究糖皮质激素受体的生物学功能奠定基础。

Objective To establish a glucocorticoid receptor （GR） gene block model in Human K562 cell with RNA interference （RNAi）technique mediated by adenovirus vector. Methods The synthetic oligonueleotide sequence, which produces RNA interference to GR, was ligated into the adenovirus shuttle vector pRNA- HI. 1 -Adeno containing green fluorescence protein（GFP） gene and homogenous recombination was conducted in the BJ5183 Escherichia coli con- taining pAdEasy - 1. The recon was transfected into 295T cells mediated by liposome and packaged to be infective adenovi- ms, which was repeatedly infected and amplified, so as to reach the appropriate titer of target cells. The adenovirus multi- ply was monitored by GFP expression. The expression of the GR protein in the infected human K562 cells was detected by Western blot. Results To construct recombination adenovirus shuttle vector psiGR/Adeno and recombination adenovirus pAdEasy- siGR successfully. GFP was expressed in recombinant adenovirus vector infected 293T cells and the expres- sions extended and brightened as time went by and appeared obvious cytopathic effect （CPE）. After 3 cycles of amplifica- tion, the adenovirus titer reached to an appropriate level. The GR protein expression in the infected human K562 cells dra- matically decreased. Conclusion The recombinant adenovirus exerting RNA interference to GR gene is successfully con- structed, laying the foundations for further research of biological function of glucocorticoid receptor.