The Effect Of Hypoxia On The Maintenance Of The Chondrogenic Phenotype In Rat Growth Plate Chondrocytes Through The HIF-1?/YAP Signal

Part one The effect of hypoxia on chondrogenic phenotype in growth plate chondrocytes in rats Objective: To study the effect of hypoxia stimulation on chondrogenic phenotype of rat growth plate chondrocytes.Method: 1.Rat growth plate chondrocytes were isolated and then stimulated using 1% oxygen concentration.2.The effect of 1% oxygen concentration hypoxia stimulation on the proliferation of growth plate chondrocytes was detected by CCK-8.3.Using RT-PCR to detect the expression of SOX9,Collagen II and Aggrecan genes in growth plate chondrocytes after 1% oxygen concentration under time gradient.4.Western blotting and immunofluorescence were used to detect the expression of SOX9,CollagenII and Aggrecan protein in growth plate chondrocytes after 1% oxygen concentration hypoxia stimulation under time gradient.Result: 1.Hypoxia has no significant effect on the growth curve of growth plate chondrocytes.2.Hypoxia promotes the up-regulation of the gene expression levels of SOX9,Collagen II and Aggrecan in a time-dependent manner.3.Hypoxia promotes the up-regulation of the protein expression levels of SOX9,Collagen II and Aggrecan in a time-dependent manner.Conclusion: Hypoxia promotes the maintenance of chondrogenic phenotype in rat growth plate chondrocytes.Part two Hypoxia promotes the maintenance of chondrogenic phenotype in rat growth plate chondrocytes by HIF-1?/YAP signalObjective: To investigate the effects of hypoxia stimulation on HIF-1? and YAP in rat growth plate chondrocytes,and to study the role and their relationship of HIF-1? and YAP in the effect of hypoxia stimulation on chondrogenic phenotype.Method: 1.The rat growth plate chondrocytes were stimulated with 1% oxygen concentration,and the expression of YAP gene,protein level and subcellular localization were detected by RT-PCR,Western blotting and immunofluorescence.2.The expressions of MST1,LATS and p-LATS under hypoxia stimulation were detected by RT-PCR and Western blotting.3.RT-PCR,Western blotting and immunofluorescence were used to detect the expression of gene and protein level,and the subcellular localization of HIF-1? and its downstream VEGF after hypoxia stimulation.4.The expression of HIF-1? was inhibited by si RNA,and then Western blotting was used to detect the expression of SOX9 protein after hypoxia stimulation.5.After si RNA was used to inhibit the expression of HIF-1?,Western blotting and immunofluorescence were used to detect the expression of YAP protein and subcellular localization after hypoxia stimulation.6.After si RNA was used to inhibit the expression of YAP,and then Western blotting was used to detect the expression of SOX9,HIF-1? and downstream VEGF protein after hypoxia stimulation.7.Reoxygenation 1 hour following hypoxia 4 hours,the expression of YAP and downstream CTGF,SOX9 and Collagen II protein levels were detected by Western blotting.Result: 1.Hypoxia promotes YAP gene level and protein level expression,and promotes YAP dephosphorylation in a time-dependent manner.Hypoxia promotes YAP nuclear translocation.2.The expression of MST1 gene and protein was increased under hypoxia stimulation,and the expression of LATS gene and protein was up-regulated,but p-LATS/LATS was significantly up-regulated after exceeded 4 hours hypoxia.3.Hypoxia up-regulates the expression of HIF-1? and its downstream VEGF gene and protein levels,and promotes nuclear translocation of HIF-1?.4.Inhibition of HIF-1? expression can significantly inhibit the up-regulation of SOX9 and YAP induced by hypoxia,and the nuclear translocation of YAP.5.Inhibition of YAP expression did not affect the expression of HIF-1?,but significantly inhibited the up-regulation of SOX9 expression induced by hypoxia.6.Reoxygenation after hypoxia can inhibit the up-regulation of YAP,CTGF,SOX9 and Collagen II protein levels induced by hypoxia.Conclusion: Hypoxia promotes the maintenance of chondrogenic phenotype in growth plate chondrocytes through HIF-1?/YAP signal.Part three Cobalt chloride promotes the maintenance of chondrogenic phenotype in growth plate chondrocytes by HIF-1?/YAPObjective: To study the effect of cobalt chloride on the chondrogenic phenotype in growth plate chondrocytes,and to study the role of HIF-1?/YAP signal in it.Method: 1.The toxic effect of cobalt chloride on growth plate chondrocytes was detected by CCK-8.2.The effect of cobalt chloride on the synthesis of extracellular matrix of growth plate chondrocytes was detected by using Alcian blue staining.3.The growth plate chondrocytes were stimulated with time gradient and concentration gradient of cobalt chloride.The expression of Collagen II and SOX9 gene levels and protein levels were detected by Western blotting and RT-PCR.4.The growth plate chondrocytes were stimulated with time gradient and concentration gradient of cobalt chloride,and then the expression of HIF-1? and its downstream VEGF were detected by Western blotting and RT-PCR.5.The growth plate chondrocytes were stimulated with time gradient and concentration gradient of cobalt chloride,and then the expression of YAP and its downstream CTGF and the subcellular localization of YAP were detected by Western blotting,RT-PCR and immunofluorescence.Result: 1.50-200?M cobalt chloride has no obvious cytotoxic effect on growth plate chondrocytes.2.Cobalt chloride can significantly promote the synthesis of extracellular matrix of growth plate chondrocytes.3.Cobalt chloride can up-regulate the protein expression of SOX9 and Collagen II in chondrocytes in a time-and concentration-dependent manner.4.Cobalt chloride can promote the up-regulation of HIF-1? and its downstream VEGF expression.5.Cobalt chloride can promote the up-regulation of YAP and its downstream CTGF expression,and the nuclear translocation of YAP.Conclusion: Cobalt chloride promotes the maintenance of chondrogenic phenotype in growth plate chondrocytes through HIF-1?/YAP...