| Background and Purpose:Digestive system cancers mainly include esophageal cancer,gastric cancer,colon cancer,rectal cancer,pancreatic cancer and liver cancer.The latest"Global Cancer Burden Report"data shows that three of the top five cancers in China are digestive system cancers(liver cancer,gastric cancer,colorectal cancer),four of the top five cancers in terms of mortality are digestive system cancer(liver cancer,gastric cancer,esophagus cancer,colorectal cancer),and there is currently no significant and effective treatment for most advanced digestive system cancers.Studies have found that natural killer(NK)cells,as an immune cell therapy method,play an important role in the treatment of digestive system cancer.In recent years,NK cell adoptive immunotherapy has made important progress in the clinical research and treatment of malignant tumors.However,due to the relatively low number of human NK cells at this stage,clinical application of treatment and scientific research trials are still subject to certain restrictions.The latest research shows that the number of NK cells and NK progenitor cells in umbilical cord blood is more abundant than that in peripheral blood and bone marrow.Therefore,this study intends to investigate the proliferation ability of umbilical cord blood NK cells cultured in small volumes in vitro and the killing efficiency of target cells,to screen the key genes of umbilical cord blood NK cell activation and explore related molecular pathways,so as to provide the possibility for adoptive immunotherapy of NK cells for digestive system tumors.theoretical basis.Methods:1.Aseptically collect 6 umbilical cord blood from healthy full-term pregnant women for lymphocyte isolation,culture in small volume for 14 days,and calculate the cell amount and determine the CD3-CD56+cell ratio by flow cytometry,the next experiment was carried out when the proportion of CD3-CD56+cells on the 14th day was>80%.2.Using the ELISA kits detect IFN-γchanges in cell supernatants of cell groups at 0 and 14 days,if the secretion of IFN-γon the 14th day was greater than 40ng/m L,the next experiment was carried out.3.Using K562-GFP as the target cell,the killing efficiency of NK cells in different groups at 0 days and 14 days was detected by flow cytometry,the next experiment was carried out when the killing rate on the 14th day was>90%.4.The 0-day and 14-day samples were sorted by CD3-CD56+cells,followed by m RNA transcriptome sequencing.5.Perform GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)enrichment analysis on the screened DEGs.6.The Gene Set Enrichment Analysis(GSEA)enrichment analysis was used to compare the pathways of DEGs that filtered out DEGs at 0 days and 14 days.7.Protein-Protein Interaction Networks(PPI)was constructed through STRING,and cytoscape is used to reconstruct and extract the core networks of interplay networks to mine key DEGs for NK activation of cord blood.Results:1.The number of NK cells in umbilical cord blood increased significantly on the 14th day(3.0±0×10~6vs 19.733±3.361×10~6)(P<0.05),and the proportion of CD3-CD56+cells on day 14(85.700±1.539%)was significantly higher than that on day0(33.167±3.014%)(P<0.05);2.When umbilical cord blood NK cells were cultured to day 14,the content of IFN-γin cell supernatant on day 14(1.370±0.451 ng/m L)was detected by ELISA method was significantly higher than the content of IFN-γat day 0(45.700±4.204ng/m L)(P<0.05);3.The flow cytometry results of NK killing experiments at 14 days of culture showed that when the effector-target ratios were 2:1,5:1,and 10:1,the killing efficiency of NK cells was significantly higher than that of NK cells cultured for 0 days(P<0.05);4.Cluster analysis and the distribution of differences in gene expression levels showed that a total of 7 524 DEGs were screened between the two groups of samples on day 0 and day 14,of which 3 970 genes were in the Up group and 3554 genes in the Down group;5.KEGG enrichment analysis showed that most of the DEGs-related pathways were significantly associated with metabolic pathways.GO enrichment analysis showed that DEGs in the Up group were located in cellular components or biogenesis,while DEGs in the Down group were mainly involved in regulating immune responses or immune system processes;6.GSEA analysis showed that the expression pathways of DEGs in NK cells on day 14 were compared with those on day 0,cell cycle,oocyte meiosis,progesterone-mediated oocyte maturation and P53signaling pathway were activated(P<0.05),complement coagulation cascade,hematopoietic cell lineage,oxidative phosphorylation,and ribosomal pathway were inhibited(P<0.05);7.10 key DEGs were screened by PPI and Cytoscape.Conclusions:1.After 14 days of culture of umbilical cord blood NK cells,the number of NK cells was significantly increased and the killing efficiency was enhanced.2.10 key DEGs(RPS27A,CDK1,RPS5,RPS13,CTNNB1,UBA52,RPS11,RPS6,RPS23 and RPS15A)related to umbilical cord blood NK activation were screened,RPS27A,RPS5,RPS13,CTNNB1,RPS11,RPS6,RPS23,RPS15A are down-regulated genes,CDK1,UBA52 are up-regulated genes. |
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