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目的探讨海藻糖低温保存血小板过程中miRNA的变化。方法收集3份来自相同血型无偿献血员的单采血小板,少量混匀后,平均分为2组。对照组:新鲜血小板组(22℃,100%血浆储存的新鲜血小板2 h);实验组:海藻糖低温保存血小板组(10℃,70%海藻糖保存液储存5 d);采用高通量测序方法对两组样品进行了Small RNA测序,进行生物信息分析,主要包括已知miRNA鉴定、miRNA差异表达分析及富集分析。结果 2组样品经Small RNA高通量测序,平均每个样品获得了11兆净读值(Clean reads),经过生物信息分析,分别获得了821个已知miRNA。通过差异表达分析,在已知的miRNA中共有460个差异表达miRNA,其中194个miRNA表达上调,266个miRNA表达下调,其中显著差异表达已知miRNA共44个,分别在钙离子通路、MAPK通路、凋亡路径、轴突导向等储存损伤相关路径中富集程度较高。相对于对照组,实验组中hsa-miR-4449、hsa-miR-1296表达显著上调(P<0.01,且差异倍数>2),hsa-miR-665表达被抑制。结论血小板经过海藻糖低温保存后的miRNA呈现出特征性表达变化,提示miRNA活动参与了低温时海藻糖对血小板储存损伤的保护作用。
Objective To investigate the changes of miRNA in trehalose during platelet preservation at low temperature. Methods Collecting three pieces of apheresis platelets from the same blood group blood donors. After a little mixing, they were divided into two groups on average. The control group: fresh platelet group (fresh platelet stored at 100 ℃ for 2 h at 22 ℃); experimental group: trehalose was stored at low temperature (10 ℃, 70% trehalose preservation solution stored for 5 days); high-throughput sequencing Methods Small RNA sequencing was performed on two groups of samples for bioinformatics analysis, including identification of known miRNAs, miRNA differential expression analysis and enrichment analysis. Results Two samples were sequenced with high throughput of Small RNA. On average, each sample obtained 11 trillion clean reads. After bioinformatics analysis, 821 known miRNAs were obtained respectively. A total of 460 differentially expressed miRNAs were found in differentially expressed miRNAs, of which 194 miRNAs were up-regulated and 266 miRNAs were down-regulated. A total of 44 miRNAs were significantly differentially expressed in the Ca2 +, MAPK , Pathways such as apoptosis, axon guidance and other storage-related pathways are enriched. Compared with the control group, hsa-miR-4449 and hsa-miR-1296 expression were significantly up-regulated (P <0.01, with a multiple of> 2), and hsa-miR-665 expression was inhibited in the experimental group. Conclusion The miRNAs of platelets after trehalose cryo-preservation showed characteristic expression changes, suggesting that miRNA activity is involved in the protective effect of trehalose on platelet storage injury at low temperature.