Research On Compression-induced Nucleus Pulposus Mesenchymal Stem Cells Apoptosis Via Mitochondrial Passway And The Correlated Mechanisms Study

Part Ⅰ Comparison of Different Methods for the Isolation and Purification of Rat Nucleus Pulposus-Derived Mesenchymal Stem CellsPurpose: Recently,nucleus pulposus-derived mesenchymal stem cells(NPMSCs)have been identified and shown a good prospect in the repair of degenerative intervertebral disc.However,there is no consensus among the methods for the isolation and purification of NPMSCs,which hinders the development of basic research and clinical application of NPMSCs.Therefore,a reliable and efficient isolation and purification method for NPMSCs is potentially needed.We aimed to compare different methods and identify an optimal method for isolating and purifying NPMSCs.Methods: NPMSCs were isolated and purified using two common methods(low density culture(LD)method and mesenchymal stem cell complete medium culture(MSC-CM)method)and two novel methods(cloning cylinder(CC)method and the combination of CC with MSC-CM method(MSC-CM+CC)).The morphology,MSC-specific surface markers(CD44,CD73,CD90,CD105,CD34 and HLA-DR),multiple-lineage differentiation potential,colony forming ability and stemness gene expressions(Oct4,Nanog,Sox2)were evaluated and compared.Results:The NPMSCs isolated from NP tissues via the four methods met the criteria stated by the International Society of Cell Therapy for MSCs,including adherent growth ability,MSC-Specific surface antigen expression and multi-lineage differentiation potential.Especially,the MSC-CM+CC method showed a relatively higher quality of NPMSCs in terms of cell surface markers,multiple-lineage differentiation potential,colony forming ability and stemness gene expressions.Conclusions: Our results indicated that NPMSCs could be obtained via all the four methods,and the MSC-CM+CC method is more reliable and efficient.The finding of this study provides an alternative option for isolating and purifying NPMSCs.Part Ⅱ Cyclosporine A attenuates compression-induced nucleus pulposus mesen-chymal stem cells apoptosis via rescuing mitochondrial dysfunction and alleviating oxidative stressAims: This study aims to investigate the protective effects and potential mechanisms of cyclosporine A(CsA),which efficiently inhibits mitochondrial permeability transition pore(MPTP)opening,on com-pression-induced apoptosis of human nucleus pulposus mesenchymal stem cells(NP-MSCs).Materials and methods: Human NP-MSCs were subjected to various periods of 1.0 MPa compression.Cell viability was evaluated using cell counting kit-8(CCK-8)assay.The cellular ultrastructure and ATP level were analyzed via transmission electron microscopy(TEM)and ATP detection kit respectively.The apoptosis ratio was determined using Annexin V/PI dual staining and terminal deoxynucleotidyl trans-ferase-mediated d UTP nick end labeling(TUNEL)assays.The levels of apoptosis-associated molecules(cleaved caspase-3,Bax and Bcl-2)were analyzed by western blot and q RT-PCR.Additionally,MPTP opening,mitochondrial membrane potential(MMP)and the levels of oxidative stress-related indicators(ROS),superoxide dismutase(SOD)and malondialdehyde(MDA)were monitored.Key findings: Annexin V/PI dual staining and detection of apoptosis-associated molecules demon-strated that compression significantly up-regulated apoptosis level of NP-MSCs in a time-dependent manner.CsA greatly down-regulated compression-mediated NP-MSC apoptosis and the cell death ratio.Compression also notably exacerbated mitochondrial dysfunction,ATP depletion and oxidative stress in NP-MSCs,all of which were rescued by CsA.Significance: Our results demonstrated that CsA efficiently inhibited compression-induced NP-MSCs apoptosis by alleviating mitochondrial dysfunction and oxidative stress.These findings provide new insights into intervertebral disc(IVD)degeneration(IVDD),and suggest CsA treatment as a potential strategy for delaying or even preventing IVDD.