Identification Of Dendritic Cells In Grass Carp Ctenopharyngodon Idellus And The Function Change Induced By Probiotic Bacillus Subtilis

Dendritic cell?DC?plays a key role in regulating host immunity.By presenting antigens to T lymphocytes,DC determines the immune response and immune tolerance and secretes a variety of immune-related cytokines to regulate the host immune activities.In this research,dendritic cell of grass carp?Ctenopharyngodon Idellus?was isolated and identified,transcriptome analysis was used to evaluate the effect of gene expression level of dendritic cell by Bacillus Subtilis Ch9,and compared the relationship between dendritic cell gene expression and host immune function,the main research contents and experiment results are as follows:Single cell suspension of spleen was isolated and cultured for 5 days,and the density gradient centrifugation method was used to obtain a kind of cell with typical dendritic morphological characteristics.The results of Transwell migration chamber experiment showed that the number of migration cells of these cells under the stimulation of LPS and Aeromonas hydrophila was significantly increased compare with the control group and the B.Subtilis treatment group.The results of the allogeneic mixed lymphatic response suggest that these cells can activate the proliferation of allogeneic lymphocytes in a dose-dependent manner.The detection of molecular surface marker CD80/86 and CD83 expression level showed that the mRNA expression levels of CD80/86 and CD83 were significantly increased after 12hours of LPS stimulation.These results indicate that these cells isolated from grass carp spleen are highly homologous in morphology and function compared with mammalian DC.UV-killed B.Subtilis Ch9 were co-cultured with dendritic cell,the total RNA was extracted at 0,4 18 h for transcriptome analysis.Using FPKM>1 as the screening criteria for gene expression,512 genes were uniquely expressed in the control group,1419 genes were uniquely expressed in the EG4 h group,559 genes were uniquely expressed in the 18 h group,and 361 genes were expressed in all three groups.Log₂ fold change?2 or log₂fold change?-2 and FDR?False discover rate?<0.05 were used as the criteria for screening differentially expressed genes.In the EG4h group,compared with the control group,the expression of 2,257 genes was significantly up-regulated and the expression of 1708 genes were significantly down-regulated.Compared with the control group,1201 genes in EG18 h group were significantly up-regulated and 969 genes were significantly down-regulated.Compared with EG4 h group,1,131 genes in EG18 h group were significantly up-regulated and 1,769 genes were significantly down-regulated.Analysis of Go functional annotation of differentially expressed genes revealed that the function of differentially expressed genes was mainly related to immune response,protein synthesis and transport,inflammatory response and lipid metabolism.Analysis of the KEGG signaling pathway showed that the interaction between cytokines and cytokine receptors was significantly enriched in both treatment groups?p<0.05?.The results showed that B.Subtilis may affect host immune function by regulating the expression of cytokines in dendritic cells.RT-qPCR was used to verify the differentially expressed genes,and the test results were basically consistent with the transcriptome analysis results,indicating that the transcriptome sequencing data were authentic and reliable.In summary,this study is the first to isolate and identify dendritic cells of grass carp,and the results of the morphological comparison and biological function analysis show that they are highly similar to mammalian dendritic cells.Transcriptome analysis showed that B.Subtilis can significantly up-regulate the expression of cytokine signaling pathway-related genes in DC of grass carp,activated the immune function of DC,enhanced the immune activities of the host,and promoted the ability to resist potential pathogens.