| Anti-disease breeding has been one of the most important work of the pig industry,and improving the immune capacity of pigs is to improve the pigs' disease resistance from the root causes.T lymphocyte is an important part of lymphocytes and plays a major role in immune regulation.However,related studies mostly involve multiple genes or important signaling pathways.With the rapid development of high-throughput sequencing technology in recent years,it's necessary to analyze the mechanism of T lymphocytes form the perspective of genome.CD4/CD8 T lymphocytes experience double negative?double positive and single positive three periods in the development of thymus,and different differentiation period of gene expression may be different.This study was designed to provide a reference for exploring the relevant molecular mechanisms of T cell development after single cell sequencing,analysis of the transcriptome data and digging the differentially expressed genes by collecting the fresh thymus tissue of piglets,preparing thymocyte suspension,labeling CD3,CD4,CD8 flow cytometry antibodies,and sorting four kinds of thymus CD4/CD8 T lymphocytes by flow cytometry.The concrete results are as flows:(1)The thymus tissues of piglets were collected,and then made them into cell suspension by enzyme digestion.Then sorted CD4~-CD8~-,CD4~+CD8~+,CD4~+CD8~-and CD4~-CD8~+ four kinds of T lymphocytes by flow cytometry.(2)By the single class cell sequencing,we got the transcriptome data of thymus CD4/CD8 T lymphocytes and found that there were a large number of differentially expressed genes in DN-DP,DP-CD4 SP,DP-CD8 SP developmental stages.(3)During the analysis of differential gene expression according to the development of the main line,we found a lot of different genes.In DN to DP developmental stages,129 genes were significantly up-regulated,125 genes were significantly down-regulated;In DP to CD4 SP developmental stages,44 genes were significantly up-regulated,104 genes were significantly down-regulated;In DP to CD8 SP developmental stages,there were 35 genes with significantly up-regulated,76 genes were significantly down-regulated.The genes' expression at DN-DP-CD4 SP and DN-DP-CD8 SP development stages could be divided into 9 cases,and the difference in each case were different.(4)When the differences of gene expression in the DN-DP stage were not significant,there were eight identical genes were significantly down-regulated between the DP-CD4 SP stage and the DP-CD8 SP stage,SLA-DQA,PTCRA,ADM5,ID1,DCK,ARPP21,XKRX,TRIM58;there was one identical gene that was significantly up-regulated between the DP-CD4 SP stage and the DP-CD8 SP stage,IL17 R.When the differences of gene expression in the DN-DP stage were significantly up-regulated,there were 12 identical genes are significantly down-regulated between the DP-CD4 SP stage and the DP-CD8 SP stage,C1 QA,PCD1B,CD1 D,EPCAM,GSX2,COL5A2,FABP6,CD1 A,RAG1,RORC,BST1,C17(f67),CD8 A and CD8 B are significanlyt down-regulated in the DP-CD4 SP stage,CD4 are significantly down-regulated in the DP-CD8 SP stage.(5)We conducted GO enrichment analysis of the differential genes in DN-DP,DP-CD4 SP and DP-CD8 SP stages,and found that the genes were involved in the immune system process,lymphocyte activation,regulation of cell activation,antigen processing and presentation processes and other processes or functions.In this study,the differentially expressed genes of thymus CD4/CD8 four classes T lymphocytes were obtained by single class cell transcriptome analysis.Differentially expressed genes were selected at different genomic levels of T cells during different stages of differentiation which provided some data for the study of porcine CD4/CD8 T lymphocytes,and also provided some new ideas for pig immunization research. |
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