重组腺病毒介导siRNA抑制大鼠肝细胞NF-κB p65的表达

Knock-down of NF-κB p65 Expression by Recombinant Adenovirus-delivered siRNA in Rat Hepatocytes

目的构建重组腺病毒Ad-NF-κB p65-siRNA,并观察其抑制大鼠肝细胞NF-κB p65表达的效果。方法(1)筛选针对大鼠NF-κB p65特异性siRNA靶序列,设计合成其对应的双链DNA,插入到pShuttle-H1中,得到psiRNA-NF-κB p65质粒。将构建好的psiRNA-NF-κB p65质粒与腺病毒骨架载体pAdEasy-1在大肠杆菌BJ5183中进行重组,酶切鉴定、293A细胞包装、扩增,得到重组腺病毒Ad-NF-κB p65-siRNA。(2)将构建好的Ad-NF-κB p65-siRNA转染大鼠肝细胞株BRL,48h后,采用RT-PCR和Western blot检测NF-κB p65表达。结果成功地构建了重组腺病毒Ad-NF-κB p65-siRNA;Ad-NF-κB p65-siRNA转染大鼠肝细胞48h后,NF-κB的mRNA水平下降了81%,细胞内的NF-κB蛋白浓度下降了71%。结论腺病毒介导的RNAi技术体外显著地抑制大鼠肝细胞NF-κB p65的表达。

Objective To construct recombinant adenovirus containing small interfering RNA （siRNA） targeting rat NF-κB p65 mRNA （Ad-NF-κB p65-siRNA）, and to evaluate its potential of suppressing NF-κB p65 expression in rat hepatocytes in vitro. Methods （1） The specific siRNA sequence targeting rat NF-κB p65 mRNA was selected, and the homologous double-strand DNA was designed and synthesized, then the DNA was inserted into plasmid pShuttle-H1, and psiRNA- NF-κB p65 was obtained. The psiRNA-NF-κB p65 was co-transfected into the E.coli strain BJ5183 with the bone plasmid pAdEasy-1, and then Ad-NF-κB p65-siRNA was generated by homologous recombination. After being identified by restriction digest and gel electrophoresis technique, the recombinant Ad-NF-κB p65-siRNA was packaged and amplified in cells 293 A. （2） Ad-NF-κB p65- siRNA was transfected into rat hepatocytes BRL cells. The content of NF-κB p65 mRNA and protein in the cells was tested by reverse transcription-PCR and Western blot respectively 48 hours later. Results The recombinant Ad-NF-κB p65-siRNA was successfully constructed. After being infected by the Ad-NF-κB p65-siRNA, the NF-κB p65 mRNA and protein content in BRL cells was decreased by 81% and 71% compared with that in the mock infected control group. Conclusion Constructed Ad-siRNA-NF-κB p65 can efficiently decrease the NF-κB p65 expression in BRL cells in vitro.