FTH1在布鲁氏菌侵染宿主细胞中的作用

为了探究布鲁氏菌侵染宿主巨噬细胞过程中铁蛋白(FTH1)生物学功能,本研究根据FTH1蛋白核苷酸序列,分别设计4条特异性小干扰RNA(short interfering RNA,siRNA)分子,亚克隆到RNAi-Ready pSIREN-RetroQ ZsGreen载体,并转染RAW264.7巨噬细胞,实时定量PCR筛选干扰效果最优的质粒;布鲁氏菌侵染干扰前、后的RAW264.7巨噬细胞,实时定量PCR检测布鲁氏菌基因16S rRNA和细胞凋亡相关基因的mRNA表达量。结果显示:获得了4条特异性siRNA分子,经测序正确,pSIREN-C siRNA质粒对FTH1干扰效果最高可达98%。干扰后布鲁氏菌侵染抑制率为84%,凋亡相关基因的mRNA表达量5个降低,3个上升。本研究表明:FTH1基因沉默后布鲁氏菌胞内的生存能力下降;布鲁氏菌侵染可以抑制细胞凋亡发生,FTH1基因沉默后细胞凋亡升高。

Effects of FTH1 in Macrophage Infected by Brucella

To explore the biological function of ferritin heavy chain （FTH1） in Brucella infecting mouse macrophage cells RAW264.7,four specific small interfering RNA were designed according to the nucleotide of FTH1 gene and inserted into RNAi-Ready pSIREN-RetroQ ZsGreen vector in this study.The recombinant vectors were transfected into mouse macrophage cells RAW264.7.Quantitative Real-time PCR was used to screen the plasmid with optimal interference effect.We detected the mRNA expression level of 16S rRNA and cell apoptosis-related genes in Brucella before and after infected with/without interference plasmids.Four specific siRNAs were obtained and sequenced correctly.The interference effect of plasmid pSIREN-C targeting FTH1 was up to 98%.The inhibiting rate of Brucella infection was 84% and the mRNA expressions of five apoptosis-related genes were down regulated,and three were up regulated after interference of FTH1.This study showed that inhibiting the expression of FTH1 could affect the ability of Brucella infecting RAW264.7 macrophage and promote apoptosis process.