In Vitro Expansion And Activation Method Of Healthy Human NK Cells And Its Hub Gene Screening And Analysis

Background:Cancer is one of the leading causes of human death,an important obstacle to increasing life expectancy globally,and has become a major public health problem worldwide.Natural killer(NK)cell immunotherapy has good clinical application prospects in the treatment of cancer,and has gradually become a research hotspot.The use of healthy human NK cells for effective in vitro expansion and then reinfusion has become an effective treatment method and has played a major role in the treatment of malignant tumors and the emergency prevention and treatment of public health emergencies.However,how to select and optimize the factors affecting the proliferation and activation of NK cells in vitro,so as to achieve the best effect of in vitro expansion of NK cells,so far,there is no consistent conclusion.Due to the limited research on the genomic information related to NK cell activation,the research on some functional genes is still relatively weak,resulting in that many mechanisms of NK cell growth,development and function regulation in vitro have not been elucidated,and their further applications are limited.Therefore,this study aimed to optimize the NK cell culture method and explore the molecular regulation mechanism of NK cell proliferation and activation in vitro under this culture method.Methods:Peripheral blood mononuclear cells(PBMC)from healthy volunteers were isolated and cultured for 14 days by applying two NK cell culture methods(group A is the experimental group,that is,CD16 monoclonal antibody and 5 kinds of cytokines;group B is the conventional control group,that is,2 kinds of cytokines are applied)to verify NK cell phenotype and function.The expansion folds of NK cells at different time periods under the two culture methods were recorded;the ratio of NK cells at different time periods under the two culture methods and the killing ability of NK cells on K562 target cells were analyzed by flow cytometry;the quantitative efficacy of interferon gamma(IFN-γ)secretion by NK cells at different time periods under the two culture methods was detected by ELISA.The screened NK cells were subjected to magnetic-activated cell sorting(MACS),and the purified NK cells at different culture stages were obtained for transcriptome sequencing(RNA-seq)and comprehensive bioinformatics analysis,Differentially expressed genes(DEGs)of NK cells in different culture stages before,during and after amplification were screened out for Gene Set Enrichment Analysis(GSEA),Gene Ontology(GO)function and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis,and then through the Protein-protein interaction(PPI)analysis to get the hub genes,hub genes was validated by quantitative real-time polymerase chain reaction(RT-q PCR).Results:1.The results of NK cell expansion ploidy showed that on the 14th day of in vitro culture,the number of cells in group A reached(4.84±0.50)×10~7 and the number of cells in group B reached(3.37±0.48)×10~7,both of which were significantly higher than the number of cells in group B.The number of cells in group A cultured on the 10th and 14th day was statistically different from that in group B at the same period.Trypan blue staining showed that the cell viability rate was above 96%during the culture period.2.The results of NK cell phenotypic assay and killing activity assay showed that on the 14th day of in vitro culture,the proportion of NK cells CD3~-CD56~+in group A and group B reached more than 90%,and more than 80%;the killing activity of NK cells on day 10 and 14 was significantly enhanced in both groups compared with that before expansion,and group A was better than group B.With the effective target ratio from 5:1,10:1 to 20:1,the NK cells in each group had a higher effect on K562 cells.The killing activity of NK effector cells against K562 target cells was gradually increased in all groups as the effective target ratio increased from 5:1,10:1 to 20:1.3.The study of IFN-γsecretion from NK cells showed that the secretion of IFN-γfrom NK cells in both groups A and B was the highest on day 14 compared with day 0 and day10,and group A was better than group B.The comparison between the two groups was statistically significant.4.RNA-seq results showed that:The RNA concentration of the samples was>100ng/μL,the RIN~e value was>9,and the total amount was>2μg;The percentage of bases of Sample Q20 was above 95%,that of Q30 was above 90%,and that of GC was between49.49%and 51.79%.And most of clean reads were distributed in the Exon region of the genome.The sequencing results were good and could be used for subsequent bioinformatics analysis.5.The results of comprehensive bioinformatics analysis showed that:NK cell samples of the same group could be well aggregated,and the correlation coefficients R~2 of the three biological repeat samples in each group were greater than 0.9.Compared with ctrl 1 group(0 d),case1 group(10 d)differentially expressed genes(DEGs)were 6343(3386 up-regulated and 2957 down-regulated);case2 group(14 d)DEGs were 4629(2402 up-regulated and 2227 down-regulated);and the case1 group compared to the case2 group had only 15 DEGs(11 up-regulated and 4 down-regulated).The gene set enrichment analysis(GSEA)and the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis showed that these DEGs were significantly enriched mainly in metabolism-related pathways,and protein-protein interaction(PPI)network analysis finally screened to obtain NDUFB7,COX5A,NDUFA6,NDUFB8,UQCRQ,NDUFB10,NDUFB9,NDUFA8,and ATP5D,and 9 hub genes.6.The RT-q PCR results showed that the RT-q PCR validation of hub genes was basically convergent with the results of RNA-seq.Conclusion:1.In this study,we applied two different culture methods to culture and amplify NK cells in vitro,compared and analyzed the results of NK cell phenotype detection and functional validation,and found that Group A cell culture method is better than Group B,which indicated that this culture method was superior to the conventional culture method of NK cells.It not only could effectively induce PBMC to become NK cells,but also enable their massive proliferation,ensure the amplification purity,and improve the cytotoxic effect of NK cells.2.In this study,through RNA-seq and comprehensive bioinformatics analysis of purified NK cells cultured to different stages in vitro,it was found that DEGs in different groups were mainly enriched in metabolism-related pathways,and 9 hub genes were finally screened,they may be involved in the process of glucose-stimulated oxidative phosphorylation(OXPHOS)to cause changes in the number and activity of NK cells during the in vitro culture cycle.