| Background Knee Osteoarthritis(KOA)is the leading cause of disability in the elderly,associated with progressive articular cartilagedegeneration.Current research suggests that with both of mechanical force and biological stimulus,chondrocytes,extracellular matrix and subchondral bone couldn’t keep homeostasis of anabolism and catabolism,especially appearing as a degradation of articular cartilage,yet the underlying pathogenesis is unclear.POSTN(periostin)is a matricellular protein which was firstly found and expressed in mouse osteoblasts,then in various human tissues.It originally mediates osteoblastic functions as well as aggregation and differentiation of prosomal cells in the periosteum.Some studies have indicated that the POSTN mRNA significantly upregulated in articular cartilage during KOA,suggesting POSTN mRNA was closely related to the development of KOA.Thus,we speculate that there may be POSTN in articular cartilage and knee joint synovial fluid,and the change of concentration of POSTN may be related to the development of KOA.The mainly objective of this study was to investigate the levels of POSTN protein in cartilage and synovial fluid during different stages of KOA.Then for further discovering the mechanisms of POSTN in cartilage,different treatments would be used in human knee cartilage cells in vitro.Finally,we used POSTN protein to intervene the surgery-induced KOA in rats and combined with observing the degeneration of its knee joint.Objective 1.To observe the effect of expression of POSTN protein on the occurrence and progression of knee osteoarthritis in the cartilage tissue and synovial fluid.2.To observe the effect of POSTN pretein on chondrocytes and further explore the molecular mechanism.3.To compare the treatment effects of the POSTN protein in a surgery-induced osteoarthritis model in rats.Methods 1.Expression of POSTN protein in cartilage and synovial fluid samples were analyzed with western blot,and enzyme-linked immunosorbent assay(ELISA).2.Cell experiment in vitro:(1)Experimental evaluation of POSTN protein positive cellualr protection on chondrocyte a.POSTN,PBS and anti-POSTN antibody were added to the culture medium.b.lipo2000 carrying POSTN mRNA was used to transfer chondrocytes,empty vector as control.c.The alginate + cartilage cell discs were cultured under Flexcell pressure,and a non pressurized group as control.d.Anti-POSTN antibody,anti-POSTN antibody + inhibitor of Wnt-β pathway and PBS were added in the culture medium.(2)Experimental evaluation of POSTN protein on proliferation of chondrocytes from tibial plateau a.POSTN,PBS and anti-POSTN antibody were added to the culture medium.The proliferations of the three groups at 24 h,48h and 72 h were determined by EDU method.b.The expression of Notch was assayed by RT-PCR,including receptors and ligands of Notch signaling pathways.c.Female 20-month-old Sprague-Dawley rats were randomly divided into 3 groups: injecting-POSTN protein group,injecting-PBS group and injecting-POSTN antibody group.At 12 weeks after the operation,related reagents were injected 3 times consecutively at 1d,3d,5d and EDU was injected into articular joint at 1day.At 2 weeks after injection,the rats were killed and the knee tibial plateau was taken to observe the proliferation of the cartilage cells.d.To observe the expression of POSTN protein in tibial epiphyseal cartilage cells by immunohistochemical technique in SD newborn mice(0-week old),1-week old and 2-week old.The tibial epiphyseal cartilage cells of SD rats were isolated in vitro and cultured to the third generation.The cells were divided into 3 groups: culture fluid+POSTN group,culture fluid+PBS group and culture fluid+ anti-POSTN group.To detect the 72 h proliferation rate of cartilage cells by EDU,and to observe the expression of Notch1 by immunohistochemical technique and western-blot.(3)Experimental evaluation of POSTN protein at the early stage of chondrocyte apoptosis a.To isolate and culture the cartilage cells of the tibial plateau of knee in vitro.The experimental group cartilage chondrocytes were divided into 3 subgroups,in which periostin,PBS and anti-periostin were added in culture medium respectively.At 24 h,48h and 72 h,the early apoptosis rate of chondrocytes was observed by flow cytometry(AV-PI)and was compared with the control group.b.At 24 h,48h and 72 h,to test POSTN mRNA and iNOS mRNA by PCR technology,to test the expression of POSTN protein and iNOS protein by immunofluorescence technique.(4)Experimental evaluation of POSTN on chondrocyte genetic phenotypes a.Effect of POSTN mRNA expression impacting the differentiation of chondrocyte phenotype b.Effect of POSTN protein on gene transcriptional regulation of chondrocyte 3.Animal experiment part:(1)To make the surgically-induced OA rat model: 60 SD rats,divided into two groups,the sham operation group only implemented slit suture,and the OA model group randomly produced one side anterior cruciate ligament transection model.Expression of POSTN protein of synovial fluid were analysed at 1,2,4,8,12 week both two groups.(2)Furthermore,we detected and compared the treatment effects of the POSTN protein in a surgery-induced osteoarthritis model in rats.Anterior cruciate ligament transection(ACLT)was performed on the random knee of rats to induce OA.Male 3-month-old Sprague-Dawley rats were randomly divided into five groups(n = 18/per group):(1)Sham + saline.(2)Surgery+ saline.(3)Surgery + Low consentration POSTN protein.(4)Surgery + High consentration POSTN protein.(5)Sergury+Antibody of POSTN protein.Intraarticular injections were performed after surgery on 3 days,1 week,2 week and 4week in the surgery knee.In vivo effects on cartilage degeneration and MMP3,13 concentration were evaluated in all rats at 4 weeks after surgery by using FMT.Gross morphologic lesions on the tibial plateau in rats were visualized by X ray,India ink staining,safranin O staining,cartilage permeation test,and OA-related protein,such as POSTN,collagen Ⅱ,MMP13,were quantified by immunohistochemistry.Results 1.Expression of POSTN protein in cartilage and synovial fluid were lower in late-stage OA than that from normal and early-stage(p<0.05).2.Cell experiment in vitro:(1)Experimental evaluation of POSTN protein positive cellualr protection on chondrocyte a.The addition of POSTN protein in culture medium could promote the proliferation of chondrocytes,increase the expression of type II collagen and decrease the expression of β-catenin(p<0.05).b.Overexpression of POSTN protein in chondrocytes could increase the proliferation of chondrocytes,promote the expression of type II collagen and decrease the expression of β-catenin(p<0.05).c.The chondrocytes were cultured under pressure,the proliferation of chondrocytes was decreased,the expression of POSTN was increased,the expression of type II collagen was decreased,and the expression of β-catenin was increased than that in non pressurized group(p<0.05),and the protein transferred from the cell membrane to the nucleus d.Inhibitor of Wnt-β pathway could reduce the negative effect of anti-POSTN antibody on chondrocytes culture(p<0.05).(2)Experimental evaluation of POSTN protein on proliferation of chondrocytes from tibial plateau a.At 24 h,the proliferation rate of POSTN group and PBS group was higher than that of POSTN antibody group(p<0.05).At 48 h,the proliferation rate of POSTN group was higher than that of the other groups(p<0.05).At 72 h,the proliferation rate of POSTN group was the highest one,the proliferation rate of PBS group was medium,and the POSTN antibody group was the lowest one(p<0.05).b.Only receptor Notch1 and ligand Jagged1 were expressed in chondrocyte.c.In rats,the proliferation rate of the chondrocytes in the medial tibia plateau of the knee of injecting-POSTN protein group was the highest,and that of injecting-POSTN antibody group was the lowest(p<0.05).d.In SD milk rats,there was a small amount of expression of POSTN protein in the static region of tibial epiphysis,a large number of expression in the proliferation area and a small amount of expression in hypercalcification area.In SD juvenile rats,there was no expression of POSTN in the static region of tibial epiphysis,a large number of expression in the proliferation area and a small amount of expression in hypercalcification area.The 72 h proliferation rate of cartilage cells was highest in culture fluid+POSTN group(p<0.05),and there was no difference between culture fluid+PBS group and culture fluid+ anti-POSTN group(p>0.05).The results of immunohistochemistry and WB showed that the expression of Notch1 in the epiphyseal cartilage cells in culture fluid+POSTN group was more than that of the other two groups(p<0.05).(3)Experimental evaluation of POSTN protein at the early stage of chondrocyte apoptosis a.The early apoptosis rate of chondrocytes with POSTN protein in medium at 6h,12 h and 24 h were lower than that of two other subgroups(p<0.05).b.At 24 h and 48 h,the relative expression of periostin mRNA and iNOS mRNA of cartilage chondrocytes in experimental group was higher than that of control group(P<0.05).At 72 h,there was no significant difference in the relative expression of periostin mRNA between the experimental group and the control group(p>0.05),but the relative expression of iNOS mRNA in the experimental group was significantly higher than that in the control group.At 24 h and 48 h,the fluorescence intensity of periostin protein in chondrocytes of experimental group was stronger than that of control group.However,the fluorescence intensity of iNOS protein in the experimental group was not significantly different from that in the control group.At 72 h,the fluorescence intensity of periostin protein in the experimental group was the same as that in the control group,but the fluorescence intensity of iNOS protein in the experimental group was significantly higher than that in the control group.(4)Experimental evaluation of POSTN on chondrocyte genetic phenotypes a.Overexpression of POSTN mRNA can promote the expression of Aggrecan mRNA,Collagen ⅡmRNA.Knockdown expression of POSTN mRNA can promote the expression of runx2 mRNA,CollagenⅩmRNA,MMP13 mRNA.b.POSTN protein enters the nucleus and regulates the transcription of CollagenⅡ gene.3.Animal experiment part:(1)There was no statistically significant difference in control group at different time stage of POSTN protein in synovial fluid(p<0.05).The POSTN protein in the intraarticular increased at first and then decreased with the development of OA.(2)FMT revealed that the knee joint treated with POSTN protein was mild in chronic inflammatory reponse at 4 weeks after surgery.Micro X ray,India ink staining,safranin O staining,cartilage permeation test,similar result were found for the the cartilage degeneration in all tests,the result was supplemental intraarticular injection of POSTN protein delayed cartilage degeneration in a rat model of OA.On the contrary,injection of POSTN protein antibody would accelerate the degeneration of knee cartilage.Conclusions 1.The expression of POSTN protein in human cartilage tissue and synovial fluid were low in normal knee joint,increased in early and middle stages,and decreased in late stage.2.POSTN protein had a positive protective effect on human knee chondrocyte.POSTN protein could promote the proliferation of human knee chondrocyte.POSTN protein could inhibit the apoptosis of human knee chondrocyte.POSTN protein maintained the phenotype of human knee chondrocyte and could enter the nucleus to regulate the transcription of Collagen ⅡDNA.3.POSTN protein was associated with the proliferation of tibia epiphyseal cartilage in rats.4.Supplemental intraarticular injection of POSTN protein delayed cartilage degeneration in a rat model of OA,suggesting that it may be a protection method for OA cartilage.Increased POSTN protein was associated with the occurrence of knee OA and decreased POSTN protein was associated with the severity of OA cartilage damage.5.POSTN protein is expected to be a biomarker for early diagnosis and treatment of knee osteoarthritis. |
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