| Research background:Crohn’s disease(CD)is an inflammatory bowel disease.At present,there is no cure for CD.Many patients have complications and require surgical treatment.The postoperative recurrence rate is very high.The exact etiology and pathogenesis of CD are not completely revealed,but the evidence shows that this disease may be caused by the interaction of multiple factors,including environmental,genetic,infection and immune factors.Intestinal macrophage dysfunction is now widely considered to be an important part of the pathogenesis of inflammatory bowel diseases.The intestine contains a large number of monocyte-macrophages,which are essential for maintaining the steady state of the mucosa on the surface of the microbiota and continuous epithelial renewal.These cells may have a role on maintaining the homeostasis of the intestinal epithelial microenvironment,but their changes and the underlying mechanism remains unknown.Research purpose and significance:Single-cell transcriptome sequencing was performed on the peripheral blood mononuclear cell(PBMC),diseased terminal ileal tissue and normal terminal ileal tissue samples from patients with CD so as to obtain single-cell-level gene expression profiles,and to obtain the patterns of individual cell subgroups of monocytes-macrophages with a preliminary molecular message.It is helpful to deepen the understand of the role of various subgroups of monocyte-macrophages in maintaining homeostasis in vivo and their changes when a protective immune response or inflammation occurs.It is also to provides a reliable basis for the future research on disclosure of the molecular mechanism on how the monocytes-macrophages contributing to CD,and of the specific marker for control of the disease.The overall goal is to clarify its pathogenic mechanism,so as to provide a theoretical basis for precise treatment and drug guidance at the molecular level.Research methods:In this study,single-cell suspensions were prepared from diseased terminal ileal tissue,normal terminal ileal tissue and PBMC samples of patients with CD.Their monocyte-macrophages were enriched by magnetic beads sorting with CD 14 antibody,and their single cell transcriptome sequencing(scRNA-seq)data was obtained on 10x Genomics platform.At present,11 samples from three CD patients(three samples each)and 1 healthy control(peripheral blood and ileum tissue)have been sequenced.Here a preliminary clustering gene expression profile,KEGG pathway and development trajectory analyses were reported on the 3 samples of monocyte-macrophages from 1 of the 3 patients.Research results:1)The activity of the single cell suspension of the peripheral blood sample is above 98%,and the activity of the tissue single cell suspension is 73%-89%;2)The preliminary quality analysis of the original data showed that the number of cells measured in the 3 samples was above 10,000,with the parameters:median genes per cell>1,000,valid barcode>90%,Q30 bases in RNA read>85%,fraction reads>70%.3)The number of genes after quality control was above 20,000,and the number of cells after removing dead cells was above 8,000.4)Through a comprehensive analysis of the monocyte-macrophages in the 3 tissue samples,14 subgroups of single cells were obtained.The monocyte-macrophage subgroups#0,#1,#2,#8,#10 and#13 were unique for PBMC sample;the subgroup#12 was unique for the diseased terminal ileal tissue sample.Except the subgroups#12,the diseased terminal ileal tissue and the normal terminal ileal tissue samples shared the same types of monocyte-macrophage subgroups,but the proportions of cells in each subgroup were different:the proportion of the monocyte-macrophages in the subgroups#5 and#11 were decreased in the diseased terminal ileal tissue sample significantly,and the proportion of subgroups#6 and#9 were increased significantly in the diseased terminal ileal tissue sample.5)Gene NR1H3 is highly expressed in subgroups 5 and 11.Research conclusions:1)Our study has successfully established a set of methods for isolation and enrichment of monocyte-macrophage single cell suspensions from terminal ileal tissue samples and PBMCs.2)The qualification analysis of the sequencing data shows that our scRNA-seq technology based on single cell suspension is succeeded.3)Preliminary analysis shows that the type,molecular pathway,development trajectory and the proportion of monocyte-macrophages are different in each of the 3 samples.Particularly compared with normal terminal ileal tissue sample,the diseased terminal ileal tissue sample have a specific pattern of subgroup of monocyte-macrophage with certain molecular profiles.4)In summary,our preliminary study suggested that monocyte-macrophages have a regulation effect on the diseased terminal ileal tissues of CD.This report is a preliminary analysis result.A further in-depth and systematic data analysis and validation research are still in progress at present. |
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