CAJ | 학술논문

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。
위료탐구포로씨균침염숙주거서세포과정중철단백（FTH1）생물학공능，본연구근거 FTH1단백핵감산서렬，분별설계4조특이성소간우 RNA （short interfering RNA，siRNA）분자，아극륭도 RNAi-Ready pSIREN-RetroQ ZsGreen재체，병전염 RAW264.7거서세포，실시정량PCR사선간우효과최우적질립；포로씨균침염간우전、후적 RAW264.7거서세포，실시정량 PCR 검측포로씨균기인16S rRNA 화세포조망상관기인적 mRNA 표체량。결과현시：획득료4조특이성siRNA분자，경측서정학，pSIREN-C siRNA 질립대 FTH1간우효과최고가체98%。간우후포로씨균침염억제솔위84%，조망상관기인적 mRNA 표체량5개강저，3개상승。본연구표명： FTH1기인침묵후포로씨균포내적생존능력하강；포로씨균침염가이억제세포조망발생，FTH1기인침묵후세포조망승고。
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.