目的:输血是住院患者常用的治疗方法。红细胞(RBC)单位在储存期间经历各种生化和形态学变化(储存损伤)。miRNA已经在细胞代谢过程中进行了深入的研究,但miRNAs对血液储存的影响尚不明确。
方法:我们基于R语言对红细胞miRNA表达的公开数据集进行了生物信息学分析,并对差异表达miRNA的靶基因进行了京都基因和基因组百科全书(KEGG)富集分析。通过qRT-PCR验证在不同时间储存的血液样品中miRNA差异基因的表达。接下来,我们用ELISA和qRT-PCR来验证IL-1β的表达,第1天和第42天血液中的IL-6、IL-12和TNF-α。此外,在体外,我们用过表达的miRNA转染巨噬细胞,流式细胞术、qRT-PCR和ELISA验证了过表达miRNA对巨噬细胞极化和炎症因子释放的影响。
结果:这项研究结合了生物信息学分析和实验,以发现长期储存的血液中差异表达的miRNA。结果显示,与新鲜血液样本相比,通过ELISA,炎症因子显著增加了一倍,以及在42天时更高的mRNA表达。实验验证miR-33a-5p通过PPARα/ACC2/AMPK/CPT-1a轴调控促进M1型巨噬细胞极化并增加相关炎症因子的释放。
结论:这项研究阐明了长期储存血液中炎症因子积累的潜在机制,为预防输血相关不良反应提供理论依据和潜在目标。
OBJECTIVE: Blood transfusion is a common therapeutic procedure in hospitalized patients. Red blood cell (RBC) units undergo various biochemical and morphological changes during storage (storage lesion). miRNAs have been studied intensively regarding cellular metabolic processes, but the effect of miRNAs on blood storage is not well defined.
METHODS: We performed bioinformatics analysis on the public data set of
miRNA expression of RBC based on R language, and performed the Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis on the target genes of differentially expressed
miRNA. The expression of
miRNA differential genes in blood samples stored at different times was verified by qRT-PCR. Next, we used ELISA and qRT-PCR to verify the expression of IL-1β, IL-6, IL-12 and TNF-α in blood at day 1 and day 42. In addition, in vitro, we transfected macrophages with overexpressed
miRNA, and the effects of overexpressed
miRNA on macrophage polarization and the release of inflammatory factors were verified by flow cytometry and qRT-PCR and ELISA.
RESULTS: This study combined bioinformatics analysis and experiments to discover the differentially expressed miRNAs in long-term stored blood. The results showed that compared to fresh blood samples, the inflammatory factors were significantly doubled by ELISA, as well as the higher mRNA expression at 42 day. Experimentally verified that miR-33a-5p promoted the M1 type macrophage polarization and increased the release of related inflammatory factors through PPARα/ACC2/AMPK/CPT-1a axis regulation.
CONCLUSIONS: This study elucidates a potential mechanism of inflammatory factor accumulation in long-term stored blood, providing a theoretical basis and a potential target to prevent transfusion-related adverse reactions.