| Temporomandibular disorder(TMD)is a disease with high incidence.Its main clinical feature is chronic pain,which is the general name of a class of diseases.OA is the main cause of joint pain and dysfunction in TMD patients.At present,the etiology of OA has not been determined,but its main pathological change is persistent inflammation.Studies have shown that chondrocytes are one of the main mediators of pathological changes in OA.Under the action of stimulation factors,chondrocytes produce a variety of cytokines and proteases to form a specific inflammatory microenvironment,which leads to the death of chondrocytes,degradation of cartilage matrix and aggravation of the development of OA.It is suggested that relevant treatment methods,if they can inhibit inflammatory reaction of chondrocytes,may prevent OA from exacerbating to explosive inflammatory stage.OA has seriously affected the quality of life of the population.However,the current treatment methods are relatively limited and cannot effectively treat OA.In recent years,based on tissue engineering methods,new strategies have been provided for the treatment of OA.Exo is a small vesicle secreted by living cells,containing cell-specific proteins,nucleic acids,lipids and other biologically active substances.Exosomes can be used as transmitters to transfer these components to receptor cells,thus regulating various physiological and pathological processes such as immune response and antigen presentation of target cells.Studies have found that exosomes derived from deciduous dental pulp stem cells have therapeutic effects on cardiovascular system,nervous system and other diseases,and studies have found that they play an important role in inhibiting inflammatory reactions such as brain injury and kidney injury.Among the biomolecules carried by exosomes,miRNA can regulate gene expression,which has attracted extensive attention from scholars.Exosomes have precise and efficient mechanism of action,and they are easy to preserve,so the targeted therapy technology of exosomes-miRNA has extremely high transformation application prospect.MiRNA in exosomes can be detected and analyzed by high-throughput sequencing method,so as to screen target miRNA related to experimental design.MTOR is a highly conserved serine/threonine protein kinase.The growth and development of cartilage,the occurrence and treatment of OA are closely related to mTOR-mediated signaling pathways.At the same time,mTOR is also an important negative regulator of autophagy.Due to the negative regulation of mTOR on autophagy,inhibition of mTOR expression,i.e.inhibition of PI3 K /AKT/mTOR pathway,can activate autophagy of chondrocytes,inhibit oxidative stress and inflammatory reaction,and reduce cell death,thus achieving the purpose of treating OA.Considering the availability of SHED,based on the biological potential of SHED-Exo to inhibit inflammatory response and promote tissue repair,and in combination with the pathological state of OA inflammatory microenvironment,we speculate whether SHED-Exo can play a therapeutic role on the inflammatory state of OA by inducing cell migration,regulating inflammatory response,etc.In this study,we mainly focus on whether the specific miRNA components in SHED-Exo can enter chondrocytes through exosomes transport,inhibit the expression of mTOR in chondrocytes,thus inhibit the inflammatory reaction of chondrocytes and treat OA.Objective :This study is an in vitro cell experiment to study the specific miRNA components in exosomes derived from dental pulp stem cells of deciduous teeth,which can enter chondrocytes through the transport of exosomes and regulate the gene expression in chondrocytes,thus inhibiting the inflammatory reaction of chondrocytes and further studying its mechanism.Methods:1.Extract deciduous dental pulp stem cells from deciduous dental pulp of children,and identify them by flow cytometry,osteogenesis and adipogenic differentiation.At the same time,condylar chondrocytes were extracted from condylar process of patients with condylar fracture,and type I and type II collagen immunofluorescence detection was carried out to identify chondrocytes.The exosomes of deciduous dental pulp stem cells were extracted by ultracentrifugation,and the surface markers CD9,CD63 and TSG101 were detected by WB method.The morphology of exosomes was analyzed by transmission electron microscope,and the particle size was detected by NTA.2.SHED-Exo was stained by PKH67 to trace and locate the entry of exosomes into chondrocytes.The effect of SHED-Exo on the migration and proliferation of chondrocytes was detected by scratch test and CCK8 cell proliferation test.In this experiment,IL-1β was used to construct a cellular model of chondrocyte OA inflammation,and the ability of SHED-Exo to inhibit chondrocyte inflammation was evaluated by detecting the expression of OA-related inflammatory factors.3.The expression of miRNA in SHED-Exo is detected and analyzed through high-throughput sequencing technology and biological information analysis of data.Combined with the expression abundance of miRNA in exosomes and bioinformatics analysis results,target miRNA(miR-100-5p)related to OA is screened according to experimental design,and whether it can be transported into chondrocytes and play a role in inhibiting inflammatory reaction(verified by miR-100-5pmimics and inhibitors)is studied.4.Through data analysis,the target gene of miR-100-5p is predicted and screened,and finally mTOR is selected as a candidate gene to study the mechanism of miR-100-5p inhibiting IL-1β induced inflammation of temporomandibular joint chondrocytes.The luciferase reporter gene activity test,WB experiment and RAPA with mTOR added to chondrocytes were carried out to explore the effect of silencing mTOR on chondrocyte inflammatory response,and the binding effect of miR-100-5p and mTOR 3’UTR was verified from various angles.Results1.The SHED extracted in this study expressed CD146,CD73 and did not express CD34.It has the differentiation potential of adipogenesis and osteogenesis and accords with the characteristics of SHED.The morphology of the extracted chondrocytes is polygon with extensive contact,and the expression of type I and II collagen is positive,which accords with the characteristics of condylar chondrocytes.The exosomes of deciduous dental pulp stem cells were extracted by ultracentrifugation.WB results showed that the expression of CD9,CD63,TSG101 surface proteins of the exosomes were significantly up-regulated compared with the SHED cell proteins of the control group.Transmission electron microscopy showed that the exosomes we extracted were small vesicles with double-layer capsular structure,showing a typical saucer type.The NTA particle size test results showed that the exosomes had uniform particle size,and the peak value(135nm)and particle size(50~150nm)were all consistent with the exosomes.2.SHED-Exo was labeled by PKH67 staining,showing that it can enter chondrocytes through endocytosis.Scratch test and CCK8 proliferation results show that compared with SHED-CM and blank control group,SHED-Exo can obviously promote cell migration and proliferation.In vitro temporomandibular joint osteoarthritis cell model was established using IL-1β group.qRT-PCR and WB results showed that compared with the control group,SHED-Exo could significantly reduce the expression of OA-related inflammatory factors.3.All miRNA in SHED-Exo were screened by high-throughput sequencing,and were sorted according to the relative content of miRNA in SHED-Exo,and the target genes of miRNA in the top 20 in SHED-Exo were analyzed by KEGG to obtain the distribution and significance of diseases and signal transduction pathways related to the target genes.After bioinformatics analysis and binding expression abundance,miR-100-5p was selected as the target miRNA in this study,and SHED-Exo was further studied to inhibit the proinflammatory induction of IL-1β in temporomandibular chondrocytes.And QRT-PCR verified that SHED-Exo can mediate miR-100-5p carried by SHED-EXO into chondrocytes.In this experiment,miR-100-5p mimic and inhibitors were transfected into chondrocytes to observe the expression of OA-related inflammatory factors,thus judging their biological functions.The results showed that overexpression of miR-100-5p significantly reduced the expression levels of MMP1,mp9 and MMP13 in chondrocytes,while silencing miR-100-5p increased the expression level of MMPs in chondrocytes,indicating that miR-100-5p has inhibitory effect on inflammatory response of chondrocytes induced by IL-1β.4.Predict and screen the target gene of miR-100-5p,and finally select mTOR as a candidate gene to study the mechanism of miR-100-5p inhibiting IL-1β induced inflammation of temporomandibular joint chondrocytes.Next,miR-100-5p luciferase reporter vector plasmids(wild type and mutant type)are constructed on the basis of pSI-Check2.The results confirm that miR-100-5p can inhibit reporter gene activity by binding to mTOR-3’UTR,indicating that the two have targeted binding.After that,WB experiment was used to detect the effect of miR-100-5p on mTOR expression at protein level.The results show that overexpression of miR-100-5p and addition of SHED-Exo can significantly inhibit mTOR expression,while inhibition of miR-100-5p expression can significantly increase mTOR expression.Finally,by adding mTOR to chondrocytes to inhibit Rapamycin,the expression level of IL-1β-induced inflammatory response related factors in temporomandibular joint chondrocytes was significantly reduced after adding Rapamycin.This indicates that mTOR can inhibit IL-1β induced inflammatory reaction of temporomandibular joint chondrocytes.It is further proved that miR-100-5p inhibits inflammatory response of temporomandibular joint chondrocytes by targeted binding of mTOR-3’UTR.Conclusion:The results show that miR-100-5p in SHED-Exo can enter chondrocytes through exosomes transport,and reduce IL-1β induced inflammatory reaction of temporomandibular joint chondrocytes by inhibiting mTOR expression,which provides new strategies and basis for OA treatment.Background: The present study aimed to validate the Centrality of Pain Scale(COPS)for use in patients with painful temporomandibular disorders(TMD).Methods: In total,166 patients with TMD were recruited to complete the Chinese translation of the COPS(COPS-C).In addition to the COPS-C,the patients were also administered the Pain catastrophizing scale(PCS)and the Pain Self-Efficacy Questionnaire(PSEQ).The reliability of the COPS-C was evaluated using internal consistency and test-retest methods.Construct validity of the COPS-C was evaluated using the exploratory factor analysis(EFA).Convergent validity was determined by analyzing the correlation between COPS-C scores and the scores of the PCS and PSEQ.Results: The Cronbach’s alpha for the total COPS-C score was 0.942.The inter-item correlations ranged from 0.356 to 0.901.The ICC values of the COPS-C ranged between 0.815-0.929.The results of EFA indicated a one-factor solution for the measure,accounting for 70.4% of the total observed variance.The factor loadings of all items ranged from 0.713 to 0.917.As for convergent validity,the COPS-C had moderate correlation with the PCS and the PSEQ.Conclusion: The results provide initial evidence that the COPS-C to be a reliable and valid measure.It can be used as a suitable instrument for pain research. |
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