| Intervertebral disc degeneration (IVDD) is the precondition andpathological basis for many degenerative spinal diseases. IVDD isgenerally considered as an age-related and multifactor-mediatedcomprehensive pathologic process. In the clinical practice, availableintervening means for IVDD and the curative effects are very limited. Anew study hotspot is to delay its degeneration by transfecting IVD cellswith exogenous genes. Especially, SIRT1has exhibited positive curativeeffects on some age-related diseases. Our studies mainly include three parts:Part I In vitro culture of nucleus pulposus cells and the study of its cellbiology charateristics; Part II Resveratrol stimulate extracellular matrixsynthesis in degenerative nucleus pulposus cells; Part III Regulation ofearly degenerative intervertebral disc nucleus pulposus cells extracellularmatrix synthesis and apoptosis by SIRT1and its signal transductionmechanism.PART IIN VITRO CULTURE OF NUCLEUS PULPOSUS CELLSAND THE STUDY OF ITS CELL BIOLOGYCHARATERISTICSObjective: To culture the nucleus pulposus cells from surgerypatienets in vitro and investigate their biological features. Methods: The NP tissues were isolated under sterile circumstance anddigested by trypsase and type II collagenase, which were continuousmonolayer cultured and identificated. NPCs at passages1,3,5, and7in thein-vitro monolayer culture were harvested to observe the morphology, cellaging, and proteoglcan expression. The cell proliferation rates andproteoglcan expression of NPCs at passage1in-vitro in monolayer cultureand in3-D alginate microsphere culture were detected.Results: The cultured cells were identified as NPCs. Morphologicalobservation, SA-β-gal and toluidine blue o staining showed thatdedifferentiation of normal NPCs tended to occur under continuous in-vitromonolayer culture, which was more obvious with increase of passagenumber. NPCs in3-D alginate microsphere culture showed significantlylower proliferation rate than NPCs in the in-vitro monolayer culture, but itcould significantly improve the protein expressions of collagen type II andAggrecan in dedifferentiated NPCs,Conclusion: The way of isolating and culturing NP cells bycontinuous enzyme digestion is successful. Continuous in-vitro monolayerculture could efficiently cultivate numerous seeding NPCs, but it is liable todedifferentiate. In3-D alginate microsphere culture restore the phenotypeof dedifferentiated NPCs and synthesize more extracellular matrix，which ismore appropriate for the futher research. PART IIRESVERATROL STIMULATE EXTRACELLULARMATRIX SYNTHESIS IN DEGENERATIVE NUCLEUSPULPOSUS CELLSObjective: To explore the impact of resveratrol on extracellularmatrix synthesis of the degenerative nucleus pulposus cells.Methods: DNPCs were derived from young patients with lumbar dischernia, which were placed in alginate culture. Primary cells in alginateculture were treated with either50μM,10μM RES or10μM,1μM sirtinolrespectively. Then mRNA expression levels of SIRT1,type Ⅱcollagen andaggrecan were detected by RT-PCR or qRT-PCR. Further stimulated withIL-1β, elevant metabolic enzymes (including MMP-2,13, ADAMTS-5, andTIMP-1) were detected by using qRT-PCR.Results: After treatment with resveratrol, the expressions of SIRT1,collagen II, aggrecan and TIMP-1mRNA were significantly increased,while the expressions of MMP-2,13, and ADAMTS-5were significantlyreduced. The results in the sirtinol group were the opposite.Conclusion: RES, as a SIRT1activator, was able to stimulate ECMsynthesis in DNPCs and protect the IL-1β-mediated ECM degradation. PART IIIREGULATION OF EARLY DEGENERATIVEINTERVERTEBRAL DISC NUCLEUS PULPOSUS CELLSEXTRACELLULAR MATRIX SYNTHESIS ANDAPOPTOSIS BY SIRT1AND ITS SIGNALTRANSDUCTION MECHANISMObjective: To study the regulatory mechanisms of SIRT1on extracellular matrix and cell apoptosis of nucleus pulposus cells duringearly intervertebral disc degeneration.Methods: Nucleus pulposus tissues were intraoperatively collectedfrom patients with lumbar disc herniation. The relations of SIRT1withECM and cell apoptosis were studied by using magnetic resonance image,immunohistochemistry, TUNEL staining, P53and P21proteindetermination, and toluidine blue staining. NPCs from the young groupwere3D-cultured with alginate in vitro, NPCs were transfected separatelyby small interfering RNA (siRNA) and lentivirus-mediatedSIRT1-overexpressed carrier. The silence and overexpression of SIRT1were target-regulated to detect its relations with ECM synthesis andapoptosis, and to find its regulatory effect on NF-κB.Results: SIRT1expression, cell apoptosis rate, and ECM synthesisability were all significantly lower in the old group than in the young group.After the expression of SIRT1was target-silenced by siRNA, thepromoting effect of resveratrol on ECM was obviously reduced. Thetransfection by lentivirus could improve SIRT1expression, protect theIL-1β-mediated ECM degradation, obviously inhibit AP, but not changeECM expression. SIRT1also showed apparent regulatory effect on NF-κB.Conclusion: SIRT1showed important regulatory effect on nucleuspulposus tissues during early degeneration, and is a potential target intreatment of intervertebral disc degenerative diseases. |
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