Using Comparative Transcriptomes To Reveal Gene Expression Changes Of T Cells During Thymus Development In Porcine

In order to reduce the economic losses caused by pig disease to the livestock industry and cultivate meat products that consumers can safely eat,animal husbandry workers should strive to increase the resistance of pigs to pathogens from the genetic nature.T lymphocytes are important components of the body to resist and eliminate pathogens,and its normal differentiation and development in the thymus is an important prerequisite for the normal function of the body's immune system.Most of the previous studies were based on single genes or single important pathways,and there are few studies having explored the differential gene expression patterns of T lymphocytes in the thymus during differentiation and development from the perspective of whole genomes.In this study,flow cytometry was used to sort 1000 CD4~-CD8~-dobule negative cells(DN cells)and two types of single positive cells(CD4~+SP cells and CD8~+SP cells)in the lymph nodes of pig intestine,followed by microarray amplification and transcriptome sequencing.Combined with the data of the four types of T cells in the thymus tissue made by the students in the previous stage of the laboratory,the T lymphocyte TCR(T cell receptor)rearrangement was performed,and the gene expression changes in the whole biological process of positive and negative selection were obtained.law.The specific results we obtained are as follows:(1)Data on three types of T cells from mesenteric lymph node were obtained by sequencing.There were 965 genes significantly up-regulated and 913 genes down-regulated in mesenteric lymph node CD4~+cells compared with thymic CD4~+cells.Compared with CD8~+cells in mesenteric lymph nodes and thymocytes,there were 1303genes with significantly up-regulated mesangial cells and 1382 genes with significantly down-regulated genes.The mesenteric lymph node CD4~-CD8~-cells were significantly different from thymic dobule negative cells,there were 1316 genes up-regulated and 1376genes significantly down-regulated.Compared with CD8~+cells,there were 46 genes significantly up-regulated in mesenteric lymph node CD4~+cells and 137 genes significantly down-regulated.(2)To compare the overlapping relationship between two groups of differentially expressed genes in the developmental process of DN(T)-DP(T)-CD4 SP(L),and found that there were a total of 109 identical genes,of which the number of up-regulated genes was 13,including BCL7A?CCDC63?ELOVL3?CTNNA1?ETV5 and other genes.The number of genes that were down-regulated was 2,namely RORA and GSAP.Comparing the overlapping relationships among the three groups of differential genes in the complete development of DN(T)-DP(T)-CD4 SP(T)-CD4 SP(L),the results showed that there were18 identical genes,which were CD8A?ACSS3?PTCRA?MNS1?HHIP and other genes.And there were no common up-regulation or down-regulation genes.(3)Compare the overlapping relationship between two groups of differentially expressed genes in the developmental process of DN(T)-DP(T)-CD8 SP(L),and found that there were a total of 120 identical genes,of which the number of genes commonly up-regulated was 21,including GATA3?TNIP3?BCL7A?ELOVL3?LPL and other genes.The number of genes jointly reduced was 8,which were TC2N?RORA?CCL5?GZMK?CD55 and other genes.Comparing the overlapping relationships among the three groups of differentially expressed genes during the complete development of DN(T)-DP(T)-CD8SP(T)-CD8 SP(L),the results showed that there were 19 identical genes,namely CD4?NOTCH3?RAG2?NEB?CLIC2 and other genes.There was no common up-regulated gene and the down-regulated gene was GSAP.(4)Two-dimensional cluster analysis of differentially expressed genes aggregated samples into mesenteric lymph nodes and thymus tissue.Further subdivision revealed that the CD4~-CD8~-,CD4~+,and CD8~+cells in the mesenteric lymph node tissue could also be clearly separated from the corresponding cells in the thymus tissue,indicating that the expression level of the cells had undergone significant changes.(5)Analysis of differential gene GO enrichment revealed that differential genes between CD4 and CD8 homologous single positive cells in both tissues were mainly enriched in adaptive immune responses,T cell-mediated immune responses,natural killer cell activation and lymphocyte activation.The differential genes of the double negative cells in the two tissues were mainly related to the processes of T cell activation,lymphocyte activation and adaptive immunity,and the differential genes of CD4 and CD8single positive cells in the mesenteric lymph node tissue were mainly enriched in concentrated in lymphocyte migration,regulation of inflammatory response,and cytokine production involved in immune response.(6)After time course analysis,we found that there were 1674 differentially significant genes in the CD4 mainline development and there were 2623 differentially significant genes in the CD8 mainline development.After functional annotation,it was found that the differential genes of T cells during development were related to immune processes such as lymphocyte aggregation,leukocyte aggregation and T cell aggregation.After obtaining the differentially expressed genes in the differentiation and development process of T cells,the relevant key genes and important pathways can be screened out to reveal the molecular mechanism of the effects and provide some theoretical basis for disease resistance breeding of pigs.