Single Cell Transcriptome Sequencing Analysis Of Chronic Skin Ulcer

Background:Chronic ulcer tissue has an imbalance in the microenvironment including cellular and intercellular abnormalities,which can slow the skin healing process.The differences of transcriptome between single cells or different cell types can be discovered by single cell RNA sequencing.Objective:Single-cell RNA sequencing was used to analyze and explore the transcriptome of chronic skin ulcer,to find out the related mechanism of chronic ulcer tissue and provide new ideas for treatment.Methods:Patients with chronic ulcer were selected,and 1×1cm ulcer tissue was collected as the experimental group,and the same volume of normal skin tissue at the same site was used as the control group.The microfluidic platform was used for single cell sorting,and the cDNA library was constructed by GEXSCOPE? massive single-cell transcriptome kit.Finally,the sequencing was performed on Illumina platform,and the sequencing results were analyzed for cell classification,cluster differential gene analysis,enrichment analysis,etc.Results:A total of 7 tissue samples were collected,including 2 hip ulcer cases,1 hip control case,3 leg ulcer cases,and 1 leg control case.A total of 10944 cells and 131983 genes were analyzed after data screening.Nine heterogeneous cell groups were found in ulcer tissue after cell marker gene annotation.Wilcox was used to analyze the differential genes of 8 cell groups in the hip and leg ulcer group and the control group(The number of chondrocytes in the two control groups was 0 and 1 respectively).The top 200 differential genes of each cell were obtained,and the high and low expression genes of the top 10 of significance were displayed in this paper.GO and KEGG analysis was further performed on the genes with high and low expression in the top ten of significance.It was found that the differential genes in B cells mainly showed low expression in cell division,mitosis and chromosome separation,and the low expression genes in endothelial cells were mainly enriched in the PI3K-Akt signaling pathway.Enrichment of expressed genes in horniness cells mainly in peptide synthesis,cytoplasmic translation,oxidative phosphorylation process.monocytes and macrophages in the respiratory electron transport chain,aerobic,cellular respiration,mitochondrial electron transport chain coupling ATP synthesis electronic chain,oxidative phosphorylation,ATP metabolism,translation,aerobic respiration,and many other processes has significant.Neutrophils lower expression of the gene enrichment in Rapl signaling pathways,Ras signaling pathways,cAMP signaling pathways.Enrichment of expressed genes in T cell mainly in carcinogenesis in virus,endocytosis.Enrichment of genetic difference between stem cells in the oxidative phosphorylation,nervous retrograde signals,and neurodegenerative disease process.Conclusion:Qualified cDNA libraries of skin chronic ulcer and normal skin were constructed.Differentially expressed genes of 8 cell types were obtained between ulcer group and control group,including granzyme genes GZMA,GZMB and GZMH,and histone genes HIST1H3C,HIST1H2AJ and HIST1H2AH were significantly decreased in ulcer group.AL031846.1 and AC009495.3 genes that had not been studied were also found.The main enrichment pathways of differential genes include many immune-related signaling pathways such as PI3K-Akt,Ras and cAMP,as well as respiratory electron transport chain,aerobic electron transport chain,cellular respiration,coupled electron chain of mitochondrial ATP synthesis,oxidative phosphorylation,ATP metabolism and other signaling pathways related to mitochondrial function.