Objective: To investigate the changes of gene sequencing and proteomics of apheresis platelet (AP) exosomes in different storage periods and predict the function of AP exosomes in different storage periods.
Methods: Platelets at different storage periods of 0 day (D0), 3 day (D3) and 5 day (D5) were collected, exosomes were extracted with Gradient centrifugation; gene sequencing and proteomic analysis were used to analyze the exosomes, and biological functions of platelet exosomes were analyzed and predicted by bioinformatics. Liquid mass spectrometry (LMS) was used to detect the changes and function prediction of exosomes proteins. The small RNA sequencing library was prepared, and the constructed library was sequenced and bioinformatics technology was used for data analysis.
Results: AP exosome iTRAQ protein analysis showed that AP exosomes stored in D3 with 55 up-regulated proteins and 94 down-regulated proteins (P<0.05, FC<0.83 or FC>1.2), while AP exosomes stored in D5 with 292 up-regulated proteins and 53 down-regulated proteins (P<0.05, FC<0.83 or FC>1.2) as compared with D0. KEGG pathway analysis showed that the proteins were mainly involved in transport and metabolism, immune system, cancer, membrane transport and other processes. There were statistically significant differences between AP exosome miRNAs in different storage days (P<0.01). The number of miRNA up-regulated and down-regulated was 374 and 255 as compared with the number of platelet exosomes miRNA stored in D3 and D0, while that was 297 and 242 in D5 and D0, and 252 and 327 in D5 and D3, respectively. The target genes of differential platelet exosome miRNAs were analyzed by GO enrichment. Target genes of differential miRNA were mainly involved in membrane composition, mainly played molecular functions binding to proteins, and participated in biological processes of transcriptional regulation.
Conclusion: The exosome differential proteins and miRNAs in D5 are significantly different from those in the D0 of APs, and they are involved in various biological processes.
题目: 不同保存期单采血小板外泌体基因测序和蛋白组学的分析.
目的: 明确不同保存期单采血小板（AP）外泌体基因测序和蛋白组学的差异变化，预测不同保存期AP外泌体的功能。方法：。结果：。结论：。.
方法: 留取不同保存期D0、D3和D5的单采血小板，用高速离心法提取外泌体，进行基因测序和蛋白组学分析，生物信息学分析并预测血小板外泌体的生物学功能。液相质谱分析技术检测外泌体蛋白的变化和功能预测；制备小RNA测序文库，对构建好的文库进行测序和生物信息学技术数据分析.
结果: AP外泌体iTRAQ蛋白分析显示，与D0相比，D3储存的AP外泌体有55个上调蛋白和94个下调蛋白（P<0.05，FC<0.83或FC>1.2），D5储存的AP外泌体有292个上调蛋白和53个下调蛋白（P<0.05，FC<0.83或FC>1.2）。KEGG通路分析结果表明，这些蛋白主要参与转运和代谢、免疫系统、癌症以及膜转运等过程。保存D0、D3和D5 AP释放的外泌体miRNA相比具有明显的统计学差异（P<0.01），其中D3与D0的外泌体miRNA相比，上调miRNA数为374，下调miRNA数为255；保存D5与D0的外泌体miRNA相比，上调miRNA数为297，下调miRNA数为242；保存D5与D3的外泌体miRNA相比，上调miRNA数为252，下调miRNA数为327。对差异外泌体miRNA的靶基因进行GO富集分析，差异miRNA的靶基因主要参与膜的组成，发挥与蛋白结合的分子功能，并参与转录调控的生物过程.
结论: AP保存末期与初期相比外泌体差异蛋白和差异miRNA显著变化，分别参与多种的生物过程.
Keywords: apheresis platelet; exosome; gene sequencing; proteomics.