摘要
目的:利用腺病毒载体介导的RNA干扰技术,建立糖皮质激素受体基因阻断的人U937巨噬细胞模型。方法:实验于2004-06/2005-05在解放军第三军医大学新桥医院呼吸研究所实验室完成。将针对糖皮质激素受体发挥RNA干扰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-1病毒骨架的BJ5183大肠杆菌内进行同源重组;重组子通过脂质体介导转染293T细胞,并在293T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到感染靶细胞的适当滴度,通过绿色荧光表达来监控腺病毒扩增;应用Westernblot法检测U937巨噬细胞感染后糖皮质激素受体蛋白的表达。结果: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U937巨噬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RNA干扰腺病毒载体。
AIM： To establish a glucocorticoid receptor （GR） gene block model in human macrophage U937 with RNA interference technique mediated by adenovims vector.
METHODS： The experiment was conducted in the Institute of Respiration Diseases, Xinqiao Hospital, the Third Military Medical University of Chinese PLA between June 2004 and May 2005. The synthetical oligonuclcotide sequence, which could produce RNA interference to GR, was ligated into the edenovims shuttle vaetor pRNAT-H1.1/Adeno containing green fluorescence protein （GFP） gene, and homogenous recombination was conducted in the BJ5183 Escherichia coli containing pAdEasy-1. The recon was trandected into 293T cells mediated by liposome and packaged to be infective adenovims, which was repeatly infected and amplified, so as to reach the appropriate titer of target cells, The adenovirus multiply was monitored by GFP expression. The expression of the GR protein in the infected human macrophage U937 was detected by Western blot.
RESULTS： GFP was expressed in recombinant adenovims vector infected 293T cells, and the expressions extended and brightened as time went by, and appeared obvious cytopathic effect （CPE）. After 3 cycles of amplification, the adenovirus titer reached to an appropriate level. The GR protein expression in the infected U937 cells dramatically decreased.
CONCLUSION： The recombinant adenovims exerting RNA interference to GR gene is constructed successfully,
出处
《中国临床康复》
CSCD
北大核心
2006年第29期89-91,i0003,共4页
Chinese Journal of Clinical Rehabilitation
