The Role Of Mechanics Sensitive Protein YAP1 In The Degeneration Of Endplate Chondrocytes

Objective: To construct a tension-induced degeneration model of human endplate chondrocytes,solve the regulatory mechanism of the mechanically sensitive protein YAP1 in inducing degeneration of human endplate chondrocytes under circulatory tension conditions,and explore potential therapeutic strategies.Method: Collect the endplate cartilage tissues of patients with lumbar disc herniation and fractures of different ages who underwent surgery in our department,and load the human endplate chondrocytes with the FX-5000 cell stress loading device in vitro.Under different tensile strength models,with 5% elongation,0.5Hz,12h/d,10%elongation,0.5Hz,12h/d,15% elongation,0.5Hz,12h/d The phenotype of human endplate chondrocytes and the expression of YAP1 were verified by Realtime RT-PCR after the mechanical conditions,and the western blot method was used for verification again.Cell staining was used to observe cartilage secretion and immunofluorescence was used to observe the human endplate cartilage before and after mechanical stimulation.Cell phenotype;select the target YAP1 for human endplate cartilage tissues and cells for experiments,and verify the expression of human endplate cartilage under different grades by tissue Realtime RT-PCR and western blot methods;then by nucleocytoplasmic separation and immunofluorescence Observe the expression of YAP1 in and out of the nucleus;construct human endplate chondrocyte knockout and overexpression models to compare with inhibitors and agonists.Protein expression and proliferation changes are detected by western blot;immunoprecipitation experiments and double The luciferase reporter gene method was used to verify the binding effect of YAP1 on Teads transcription factor and the binding effect of YAP1 on type II collagen and proteoglycan promoter;and use human endplate cartilage tissue to verify the therapeutic effects of agonists through tissues.The changes in the levels of type II collagen and YAP1 were detected by chemical staining and immunohistochemical methods;finally,the upstream of YAP1 was verified and the Piezo protein was screened by Realtime RT-PCR,and the corresponding inhibitors were used to interfere with Piezo1,and then the changes in cartilage-related protein and YAP1 levels were observed.Results: The greater the mechanical stimulus,the weakest human endplate chondrocyte phenotype expression.Correspondingly,its expression level will gradually decrease with the increase of mechanical strength and the corresponding time.In terms of proliferation,it can be found that the mechanical strength of 10% achieves the best effect.Co-immunoprecipitation experiments in endplate chondrocytes show that YAP1 and Teads have a binding effect;after YAP1 is knocked out by inhibitors and lentivirus,the phenotypic expression of endplate chondrocytes is significantly inhibited,and the reverse agonist and lentivirus overexpress YAP1.Ascending,the dual luciferase reporter gene method was used to verify that YAP1 mainly regulates the promoter of the endplate chondrocyte phenotype,thereby acting on its gene expression;and the agonist has alleviated the degree of degeneration of the endplate cartilage tissue;finally passed the test The expression of Piezo and last1 indicate that Piezo can regulate YAP1 but not through the Hippo pathway.Conclusion: Mechanical stimulation can cause human endplate chondrocytes to degenerate,and 10% tension has a positive effect on the growth of human cartilage endplates;overexpression of YAP1 can alleviate the degree of endplate chondrocyte degeneration.