| THE ISLOLATION, EXTRACTION, CULTURE OF NUCLEUSPULPOSUS CELLS IN VITROObjective: To master the skills of islolation, extraction, culture ofnucleus pulposus (NP) cells, and to provide the enough cells for thefollowing experiments.Methods: Nucleus pulposus samples were separated, trypsinized by0.25%trypsin and0.2%collagenase type II. To identify the NP cells, theexpression of COL2A1was assayed by immunofluorescence staining. TheCOL2A1mRNA and protein of NP cells were measured after differentculture methods (frozen, non-frozen, monolayer culture,3D culture).Results: NP cells were harvested successfully, and identified by theimmunofluorescence staining. After frozen, NP cells significantly decreasedthe expression of COL2A1(P<0.05), and3D culture obviously increasedthe expression of COL2A1in the NP cells (P<0.05). Conclusion: Our methods can get NP cells successfully. The phenotypeof frozen NP cells is changed, but the phenotype of NP cells is restored after3D culture. THE EFFECT OF SIRT1INHIBITING TNF-ALPHA INDUCEDEXPRESSION OF MCP-1IN DEGENERATIVE NUCLEUSPULPOSUS CELLSObjective: To explore the effect of SIRT1regulate the expression ofMCP-1in nucleus polposus stimulated by TNF-α.Methods: The proliferative activity and expression of MCP-1/SIRT1were assayed after NP cells stimulated by TNF-αat different time (12H/24H)and concentration (5ng/ml,10ng/ml,20ng/ml). NP cells were treated byResveratrol (activator of SIRT1), Nicotinamide (inhibitor of SIR-T1),siRNA of SIRT1and (or) TNF-α, then the mRNA and protein ofMCP-1/SIRT1were assayed by qPCR and Western Blot.Results: the stimulation of TNF-α had no effects on cell proliferativeactivity at different time and concentration. The expression of MCP-1washighest when the NP cells was stimulated by TNF-α at24hours andconcentration20ng/ml (P<0.05). Pretreatment with Resveratrol decreasedMCP-1expression in NP cells induced by TNF-α, while Pretreatment withNicotinamide and siRNA of SIRT1increased MCP-1expression in NP cellsinduced by TNF-α.Conclusion: SIRT1can decrease the expression of MCP-1in nucleuspolposus stimulated by TNF-α. THE MECHANISM OF SIRT1INHIBITING TNF-ALPHAINDUCED EXPRESSION OF MCP-1IN DEGENERATIVENUCLEUS PULPOSUS CELLSObjective: To explore the molecular mechanism of SIRT1decreasedthe expression of MCP-1in nucleus polposus stimulated by TNF-α.Methods: NP cells induced by TNF-αpretreated with curcumin(inhibitor of AP-1), mRNA and protein expression of MCP-1was assayed byqPCR and Western Blot. NP cells induced by TNF-αpretreated withResveratrol, then localization and expression of c-Jun (one of AP-1subunits)was detected by immunofluorescence staining and Western Blot. pretreatedwith Resveratrol, and siRNA of SIRT1, the AP-1activity in NP cellsstimulated by TNF-αwas assayed by EMSA.Results: Pretreatment with curcumin decreased MCP-1expression inNP cells induced by TNF-α. Resveratrol decreased the expression of c-Jun innucleus (P<0.05). EMSA showed that Resveratrol decreased the activity ofAP-1and siRNA of SIRT1increased the activity of AP-1(P<0.05).Conclusion: SIRT1decrease the expression of MCP-1in nucleuspolposus stimulated by TNF-α, through AP-1signaling pathway. |
