Differential Analysis Of Normal And Degenerated Nucleus Pulposus Cells Based On Single Cell Sequencing

Objective:Through bioinformatics analysis,the single-cell transcriptome sequencing expression profiles of human normal nucleus pulposus cells(NNP)and human degenerative nucleus pulposus cells(DNP)were compared to clarify the transcriptome differential expression profiles of human normal and human degenerative nucleus pulposus cells;bioinformatics was used to find out the genes related to nucleus pulposus cell calcification,fibrosis,reversal of fibrosis,inflammation,and pain,and to explore the new cells in nucleus pulposus cells and the biological processes related to the degeneration process.Method:Two nucleus pulposus tissue samples were obtained by surgery.NNP samples were obtained from the L4/5 intervertebral disc of a patient with acute spinal cord injury who underwent open lumbar surgery,and DNP samples were obtained from the L4/5 intervertebral disc of a patient with disc degeneration who underwent open lumbar surgery.Single cell transcriptome sequencing was performed on each sample tissue.The single cell sequencing results of the two samples were analyzed using bioinformatics methods to compare the differences in the composition of human normal nucleus pulposus and human degenerated nucleus pulposus tissue cells,map the histiocytes of NNP and DNP,perform cell differentiation trajectories for the cell populations of interest and predict cell function.Results:After single-cell sequencing of human normal and human degenerated nucleus pulposus tissue samples,the sequencing results of NP samples were analyzed using bioinformatics methods,and nine cell populations were clustered and nine cell types were identified,which were Chondrocyte 1,Chondrocyte 2,Chondrocyte 3,Chondrocyte 4,Chondrocyte 5,Endothelial,Macrophage,Neutrophil,and T cells.Analysis of the proportion of chondrocytes in different tissues revealed that chondrocyte 1accounted for a higher proportion of normal nucleus pulposus tissue and highly expressed COL2A1 compared with degenerated nucleus pulposus tissue;chondrocyte 2,chondrocyte 3,chondrocyte 4,and chondrocyte 5 accounted for a higher proportion of degenerated nucleus pulposus tissue compared with normal nucleus pulposus tissue.Among them,chondrocyte 2 was an inhibitory calcified chondrocyte with high expression of MGP.Chondrocytes 3 were fibrochondrocytes with high expression of COL1A1.Chondrocytes 4 are chondrocytes that highly express pain inflammatory genes such as PTGES.Chondrocytes 5 were calcified chondrocytes with high expression of FN1.(Chondrocytes 4 and chondrocytes 5 were found for the first time in a study of single-cell transcriptome sequencing of disc tissue.)Cell trajectory analysis revealed that chondrocyte 1 was at the beginning of the trajectory and chondrocyte 3 was at the end of the trajectory,while chondrocyte 5appeared first in the trajectory relative to chondrocyte 2 and chondrocyte 4.Human normal nucleus pulposus cells treated with TNFα for different periods of time were detected by RT-q PCR for the expression levels of highly expressed specific genes: COL2A1,MGP,COL1A1,PTGES,TIMP1,and FN1 genes in different cell populations.After the addition of TNFα,COL2A1 expression decreased,COL1A1 expression increased,and PTGES expression increased,and the differential change in expression was statistically significant.MGP expression decreased and then increased,or because the gene reversed calcification function required conditions to be activated,still to be verified,the expression difference change was statistically significant.However,the expression levels of TIMP1 and FN1 genes did not reach the expected results of the experiment.Conclusion:Human normal and human degenerated nucleus pulposus tissue cell profiles can be successfully mapped by single-cell sequencing,explaining the cellular heterogeneity of human normal and human degenerated nucleus pulposus tissue during the progression of lumbar degenerative diseases.Among the nucleus pulposus histiocyte types,they can be mainly divided into chondrocyte group and non-chondrocyte group,of which chondrocyte group accounts for the main component.Through cell trajectory analysis,it was found that chondrocytes 5 specifically expressing FN1,SESN2,and GDF15 may be the key cells leading to degeneration of nucleus pulposus cells.Chondrocytes 2 expressing MGP,MT1 G,and GPX3 may play a role in reversing calcification and degeneration,and chondrocytes 4 expressing PTGES,TREM1,and TIMP1 may play a role in disc degeneration pain and inflammation.This study suggests that there are multiple chondrocyte types in nucleus pulposus tissue,and intervention of a certain chondrocyte type may delay the progression of degeneration.The results of this study can provide an important reference for studying the pathogenesis of degenerative disc disease,and deepen the understanding of chondrocyte diversity in nucleus pulposus tissue,providing a reference cell map for studying cartilage disease research.