| Part I:The expression changes and effects of Hippo signaling pathway in rat endplate chondrocyte degeneration induced by IL-1βResearch purpose1.Observe the effect of IL-1β on rat endplate chondrocytes.2.Observe the effect of Hippo signaling pathway on IL-1β induced degeneration of rat endplate chondrocytes.Research method1.The chondrocytes of male SD rats aged 6-8 weeks were extracted from the tail lamina,and the growth of the cells were observed to be more than 80%and passed into P1 generation,which were cultured for 24 hours and labeled as the control group.The other group of P1 generation cells were added with different concentrations of IL-1β(5,10,15 ng/ml),and the cells were collected and screened and labeled as the IL-1β group.2.Toluidine blue staining,CCK-8,flow cytometry,immunofluorescence,Western blot and qRT-PCR were used to observe the activity and apoptosis of endplate chondrocytes and the phenotypes of Collagen II,Sox-9 and MMP-13 stimulated by IL-1β.3.mRNA and protein levels of Hippo-YAP1 signaling pathway related molecules in endplate chondrocytes of the two groups were detected by qRT-PCR,Western blot and immunofluorescence.4.The effect of YAP1 overexpression on endplate-chondrocytes under IL-1β was observed by qRT-PCR,Western blot and immunofluorescence,respectively.Result1.Immunofluorescence showed that the positive rate of Collagen Ⅱ expression was more than 90%,indicating successful extraction of endplate chondrocytes.By CCK-8 and flow cytometry,it was found that the apoptosis level of endplate chondrocytes increased in the group with 10ng/ml IL-1β,and the activity was good for follow-up experiments.Toluidine blue staining showed that the cells in the control group were polygonal and pavel-like,and arranged closely.The IL-1β group was irregularly spindle-shaped and sparse.2.Compared with the control group,mRNA levels of Collagen II and Sox-9 in IL-1βgroup were significantly decreased,and the level of phenotypic protein and fluorescence expression were decreased,while the mRNA and protein levels of MMP-13 were increased.3.Compared with the control group,mRNA levels of MST2,LATS1/2,YAP1 and CTGF decreased in IL-1β group.Western blot and immunofluorescence tests showed that the expression of YAP1 and CTGF in the nucleus and cytoplasm of endplate cartilage decreased,while the expression of P-YAP1 increased,suggesting that the nuclear inmigration of YAP 1 decreased and thus inhibited its transcriptional activity.4.Compared with the IL-1β group,qRT-PCR,Western blot and immunofluorescence showed that the expressions of Collagen II,Sox-9 and MMP-13 increased after overexpression of YAP1.ConclusionRegulation of YAP 1 overexpression can alleviate IL-1β-induced chondrocytes degeneration in rat endplate.Part II:The role of miR-210-5p in regulating YAP1 expression in IL-1β-induced degeneration of rat endplate chondrocytes Research purposeExplore the mechanism of miR-210-5p regulating YAP1 expression in rat endplate chondrocytes degeneration induced by IL-1β.Research method1.Bioinformatics software was used to screen upstream targeted regulatory molecules of YAP1.qRT-PCR was used to detect the difference in expression of candidate molecules between control group and IL-1β group.2.Luciferase experiment verified the relationship between miR-210-5p and YAP1.When miR-210-5p was overexpressed and silenced,the phenotypes of YAP1 and endplate chondrocytes were detected by qRT-PCR,Western blot and immunofluorescence under IL-1βResult1.The analysis of bioinformatics soft ware found that miR-210-5p had the binding site of YAP1 gene with a high fraction.Compared with the control group,qRT-PCR showed increased expression of miR-210-5p in the IL-1β group,and luciferaseverified the targeted binding effect of miR-210-5p and YAP1.The expression of miR-210-5p was significantly increased under IL-1β,and the expression trend of miR-210-5p was opposite to that of YAP1 in rat endplate chondrocytes.2.Compared with the IL-1β group,mRNA levels and protein expressions of YAP1,Collagen II and Sox-9 were down-regulated after overexpression of miR-210-5p,overexpression of miR-210-5p promoted the increase of MMP-13 expression.On the contrary,the expression of YAP 1 was up-regulated after the inhibition of miR-210-5p,Collagen II and Sox-9 were down-regulated,and the expression of MMP-13 was increased.Immunofluorescence indicated that overexpression of miR-210-5p would aggravate the decrease of YAP 1 expression,the YAP1 fluorescence expression was increased after inhibition of miR-210-5p.ConclusionKnockdown of miR-210-5p alleviates IL-1β-induced chondrocytes degeneration in rat endplate by targeting YAP1.Part Ⅲ:The role of miR-210-5p/YAP1 regulatory axis on intervertebral disc degeneration in ratsResearch purposeThe effect of miR-210-5p on the degeneration of rat endplate cartilage and intervertebral disc was observed by regulating the expression of miR-210-5p in vivo.Research methodRats were randomly divided into 4 groups with 10 rats in each group and control group without intervention.The caudal vertebra was modeled with 18G needle and was labeled as degeneration group according to Pfirrmann grade of 4-5.10ul PGMLV-SC5 was injected into the coccygeal endplate cartilage labeled Ctrl+PGMLV-SC5 group.After caudal puncture,PGMLV-SC5 10ul was injected into the endplate cartilage as IDD+PGMLV-SC5 group.MRI,X-ray and tissue staining were performed 8 weeks after surgery to observe the changes of endplate cartilage and intervertebral disc,and the expressions of Collagen Ⅱ and YAP 1 in tissues were observed by immunohistochemistry.Result1.Compared with the control group,X-ray results showed obvious loss of vertebral disc height accompanied by a few osteophytes in the degenerative group,and the loss of vertebral disc height was relieved in the IDD+PGMLV-SC5 group.In the control group,MRI showed that Pfirrmann grade was 1.The Pfirrmann grade of the degeneration group was 4-5,and the endplate cartilage was blurred.The Pfirrmann grading of IDD+PGMLV-SC5 group was 3-4.2.Compared with the control group,HE and Saffron-O green staining showed that the intervertebral disc annulus were disordered,the nucleus pulposus were dehydrated,and the endplate cartilage were broken in the degenerative group.Compared with the degeneration group,the degeneration of rats in IDD+PGMLV-SC5 group was reduced.Compared with the control group,immunohistochemistry showed that the expressions of Collagen II and YAP1 in the degenerative group were lower.Compared with the degeneration group,the expressions of Collagen Ⅱ and YAP1 were higher after miR-210-5p knockdown.ConclusionInhibition of miR-210-5p expression in vivo alleviates endplate cartilage and intervertebral disc degeneration in rats.Part Ⅳ:The effect of MettL3-mediated m6A modification on miR-210-5p maturation on IL-1β-induced degeneration of rat endplate chondrocytesResearch purposeObserve and verify the regulatory effect of METTL3 mediated m6A methylation modification on the expression of miR-210-5p.Research method1.Western blot and qRT-PCR were used to detect the expression of METTL3 in IL-1β-induced endplate chondrocytes degeneration.2.Regulate METTL3 to observe the phenotype and expression changes of YAP1,miR-210-5p and pri-miR-210-5p in endplate chondrocytes under IL-1β.3.Co-immunoprecipitation further determined whether METTL3 produced targeted adhesion to miR-210-5p through DGCR8.Result1.Western blot and qRT-PCR indicated that the expression of METTL3 increased in IL-1β-induced degeneration endplate chondrocytes.After METTL3 knockdown,phenotypic inhibition was alleviated,and YAP1 mRNA expression level and fluorescence expression were increased.2.The expression of miR-210-5p decreased after METTL3 inhibition,while the expression of pri-miR-210-5p increased significantly.Co-immunoprecipitation confirmed that METTL3 was able to bind to DGCR8,a key protein for miRNA maturation,suggesting that METTL3 silencing interfered with pri-miR-210-5p cleavage.ConclusionSilencing METTL3 regulates miR-210-5p maturation by mediating m6A modification and alleviates IL-1β-induced endplate chondrocyte degeneration.Part Ⅴ:Correlation between expression of METTL3/miR-210-5p/YAPl regulatory axis and degeneration of human endplate cartilage Research purposeThe expression differences of METTL3,miR-210-5p and YAP1 in endplate cartilage were determined.Research methodTwenty cases of isthmus endplate cartilage specimens were set as the control group,and 20 cases of lumbar disc herniation specimens were set as the degeneration group.The expression differences of METTL3,miR-210-5p and YAP1 in the two groups were detected by HE staining,qRT-PCR,Western blot and immunohistochemistry,and the correlation between the three groups was analyzed.Result1.HE staining showed that the extracellular matrix of endplate cartilage in the control group was higher than that in the degeneration group.The expression of METTL3 and miR-210-5p was higher in the degenerative endplate cartilage,while the expression of YAP1 was lower.2.METTL3 was positively correlated with the expression of miR-210-5p,while YAP1 was negatively correlated with the expression of METTL3 and miR-210-5p.ConclusionCompared with the control group,the expressions of METTL3 and miR-210-5p were higher and the expression of YAP1 was lower in endplate cartilage of the degenerative group. |
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