| ObjectivesIntervertebral disc degeneration is widespread in people of all ages,and aging of nucleus pulposus cells plays a key role in clinical spinal events.Previous studies have found that mesenchymal stem cell phenotypes are compensated up-regulated in nucleus pulposus tissues with different degrees of degeneration.Limited by the local anatomy,it gradually decreases at the end stage.Previous studies have shown that mesenchymal stem cells have partial remission of aging nucleus pulposus cells and enhance their cell viability.However,the paracrine effect of stem cells triggers little protective response to nucleus pulposus cells Therefore,this study is used as an entry point to explore the effects of three-dimensional co-cultured mesenchymal stem cells on the aging of nucleus pulposus cells and the possible downstream mechanismsMethods1.To study the relationship between the changes of medullary nucleus and the clinical grading indexes of the nucleus pulposus clinical tissue specimens with different degrees of degeneration and the rat tail degeneration model2.Construct a three-dimensional co-culture system with different cell densities,and observe the alleviation and influence of mesenchymal stem cells on aging of demyelinated cells3.To study the extensive and complex changes caused by mesenchymal stem cells to nucleus pulposus cells,RNA-seq technology was used to determine the mesenchymal stem cells against the changes in cell synthesis and signaling pathways.Possible targets for nuclear cells4.Based on the results of previous studies,a stable ZMPSTE24 knockout nucleus pulposus cell line was constructed using the crispr cas9 gene editing technology for the ZMPSTE24 gene,which was significantly altered with nucleus pulposus cells.ZMPSTE24 overexpression and knockout were observed in vitro.Nuclear cell synthesis and catabolism and the effects of cell cycle status5.To further verify the changes of mesenchymal stem cells to nucleus pulposus tissue,an in vitro organ culture technique was used to observe the effect of mesenchymal stem cells on the degeneration of nucleus pulposus tissue at the organ level and whether it can alleviate the changes of nucleus pulposus degenerationResults1.The expression of CD44,one of the important phenotypes of mesenchymal stem cells,was gradually reduced in the short-term up-regulation in the rat tail-buckling model and clinical specimens with different clinical grades.The positive rate of SA-β-gal staining with the progression of tissue degeneration was gradually increased,and the difference was statistically significant2.The three-dimensional alginate gel material loaded with mesenchymal stem cells showed a salvage effect on the senescence phenotype of nucleus pulposus cells under different cell densities and co-culture time:decreased positive staining rate,increased cell activity,and cell synthesis Decompose balance reconstruction3.Co-cultured nucleus pulposus cells showed significant differences from the control group in cell structure,biological synthesis ability and biological process.Among them,the sensitization of nucleus pulposus cells was up-regulated,the extracellular matrix synthesis process was enhanced,and the inflammatory markers IL1,IL6,IL10,IL13,IL33 were down-regulated compared with the control group4.After overexpression and knockout of nucleus pulposus cells ZMPSTE24,in the condition of inflammatory induced aging,different groups of nucleus pulposus cells showed significant differences in cell aging positive rate,cell activity,cell synthesis ability and degradation of extracellular matrix.Overexpression of ZMPSTE24 can effectively reduce the positive rate of cell aging,increase the cell viability,and enhance the synthesis ability of nucleus pulposus cells under induced aging stress5.After culturing nucleus pulposus tissue in ex-vivo enviroment,the height of nucleus pulposus tissue in the bone marrow mesenchymal stem cell group was significantly lower than that in the nucleus pulposus cell co-culture group and the blank control group.The positive rate of aggrecan and Brdu immunohistochemistry in the stem cell co-culture group was significantly higher than that in the nucleus pulposus cell co-culture groupConclusionsThe correlation between the positive rate of nucleation of nucleus pulposus cells and the degree of degeneration of intervertebral discs in this study confirmed the responsiveness of mesenchymal stem cells to nucleus degeneration.The transcriptional protection of mesenchymal stem cells to nucleus pulposus cells in a co-culture system was confirmed The protective effect of the aging gene ZMPSTE24,which is associated with nucleus pulposus degeneration,on inflammatory aging stress was identified and confirmed,explaining the possible mechanisms of action with the inflammatory pathway.It also provides a theoretical basis for ZMPSTE24 as an early diagnostic marker or therapeutic target for the prevention and treatment of intervertebral disc degeneration. |
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