The Effect And Mechanism Of The MMP-1/13 Expression Activate By Poly(I:C) In Human Nucleus Pulposus

Objective:Isolating,culturing and identifying the nucleus pulposus(NP)cells to investigate the proliferation character.To explore the mechanism and signaling pathways of the MMP-1/13 expression activated by Poly(I:C)in human nucleus pulposus cell.Methods:(1)Digested and isolated nucleus pulposus cells by trypsin and type ? collagenase complex.(2)Identifying the NP cells cultured in vitro by determining the related gene expression by real-time PCR,compared to Articular Cartilage(AC).(3)Determined the proliferation of NP cells stimulated by Poly(I:C)with CCK-8kit,compared with NP cells treated with PBS.(4)Detecting the m RNA and protein expression of MMP-1/13 in NP cells stimulated by Poly(I:C)with real-time PCR and Western blot.(5)Detecting the MMP-1/13 m RNA expression of NP cells stimulated by different ligand of toll-like receptors(TLR)with real-time PCR.(6)After blocking or silencing the TLR3 signaling pathway by Hydroxychloroquine(HCQ)or Small interfering RNA(si RNA),detected the MMP-1/13 m RNA expression of NP cells stimulated by Poly(I:C)with real-time PCR.(7)Silencing the MDA-5/RIG-I signaling pathway with si RNA,detected the MMP-1/13 m RMA expression of NP cells stimulated by Poly(I:C)with real-time PCR.(8)Measuring the m RNA and protein level of inflammatory factors of NP cells stimulated by Poly(I:C)with real-time PCR and ELISA.(9)After inhibiting the inflammatory factors by Cycloheximid(CHX),detected the MMP-1/13 m RMA expression of NP cells stimulated by Poly(I:C)with real-time PCR.Results:Plenty of NP cells could be obtained after the NP tissue predigested with trypsin for 30 min and subsequently digested with collagenase ? for 4hr.Isolated NP Cells highly expressed COL2A1 and SOX9 genes.Compared with AC cells,NP cells expressed PAX1?FOXF1 and CA12 genes higher,while expression of FBLN1?GDF10and CYTL1 genes had no significant difference.The logarithmic phase of adherent growth of NP cells cultured in vitro was 48~72hr.The proliferation activity of human NP cells maintained within 8~48hr treated with6.25-50?g/ml Poly(I:C).MMP-1/3/13 m RNA of NP cells expressed increased by stimulation of Poly(I:C),while the MMP-1/13 level increased significantly.Compared with the control group,m RNA level increased in NP cells treated the corresponding ligand of TLR1-6?TLR9,while the m RNA expression had no significant difference after NP cells treated the ligands of TLR1?TLR2?TLR4?TLR5?TLR6?TLR9.The m RNA and protein of MMP-1/13 had higher expression level,compared to control group.The m RNA and protein expression of MMP-1/13 presented the dose-time independent effect with Poly(I:C),and peaked in 24 hr treated with 50?g/ml Poly(I:C).After lysosomal acidification inhibitor hydroxychloroquine(HCQ)pretreatment of NP cells by blocking TLR3 pathway,the MMP-1/13 m RNA expression decreased in Poly(I:C)stimulated group(ie,HCQ+Poly(I:C))compared with no blocking TLR3group(ie,Poly(I:C)).NP cells treated with Poly(I:C)had lower MMP-1/13 m RNA expression in silencing TLR3 group with si RNA compared with no silencing group.NP cells treated with Poly(I:C)and si RNA of MDA-5/RIG-I had no significant difference MMP-1/13 m RNA expression compared with no silencing group.Poly(I:C)stimulated NP cells expressed inflammation factor IL-6 and TNF-?.The protein synthesis inhibitor cycloheximide(CHX)could inhibit the production of IL-6and TNF-? and other inflammatory factors.NP cells with CHX pretreatment stimulated with Poly(I:C)had lower MMP-1/13 expression compared with no CHX pretreatment.Conclusion:Adequate cells could be obtained for following experiments from nucleus pulposus tissue by combination of trypsin predigested and type ? collagen enzymatic digestion.Nucleus pulposus cells were identified with marker gene expression.Nucleus pulposus cells cultured during 48-72 hr could be used in the subsequent experiments.MMP-1/13 expression of NP cells stimulated by Poly(I:C)showed significant dose-time independent effect within the appropriate dose and time.MMP-1/13 expression stimulated by Poly(I:C)is mainly dependent on TLR3 signaling pathway,rather than MDA-5/RIG-I signaling pathway.Poly(I:C)stimulates the secretion of cytokines IL-6/TNF-? of nucleus pulposus cells,and inflammatory factors might amplify the expression of MMP-1/13 in the procedure of Poly(I:C)stimulation.