Decoding the Cellular and Transcriptomic Features of Natural Killer Cells from Peripheral Blood of Severe Acute Pancreatitis

The study subjects included 19 patients with acute severe pancreatitis and 19 age- and gender-matched healthy controls (HCs). PBMCs were isolated from 5-10 mL fresh and sterile peripheral blood of HCs and patients with SAP. After that, the cells were cryopreserved or used for further analyses such as cell counting and flow cytometry (FCM) analysis of the resident NK cells in PBMCs (rHC-NKs, rSAP-NKs).

FCM was performed at the indicated time points (day 0, day 14). Cell cycle of HC-NKs and SAP-NKs was detected by FCM assay. The cytotoxicity of eHC-NKs and eSAP-NKs was evaluated using a co-culture model with specified tumor cell lines (K562, U937, Nalm6) at an effector-to-target (E: T) ratio of 1:5.

Total mRNAs were extracted from eHC-NKs and eSAP-NKs by utilizing the TRIZol reagent (ThermoFisher, USA) according to the manufacturer’s instructions. For RNA-SEQ analysis, we turned to Lianchuan (Hangzhou, China) for detection and the online Omicstudio platform (omicstudio.cn/home) for further bioinformatics analyses such as Venn Map, Gene Set Enrichment Analysis (GSEA), Volcano plots, Gene Ontology Biological Processes (GOBP), Principal Component Analysis (PCA), and Kyoto Encyclopedia of Genes and Genomes (KEGG).