The Establishment Of Human Senescence Intervertebral Disc Nucleus Pulposus Cells Model And The Preliminary Research On The Senesence Cell Phenotype

Obiective:To establish the human senescence interbertebral disc nucleus pulposus cells(NPCs) model and investigate the differential expression of growth factors in normal and senescence intervertebral disc NPCs, and discuss the relationship between these growth factors and intervertebral disc degeneration.Methods:1. Normal NPCs(Scien cell 4800) were monolayer cultured in vitro, continous subcultured under 1:2, then the replication senescence cells (RS) model was set up;2. Normal NPCs were forced on different concentration of hydrogen peroxide, then the stress induced premature senescence cells(SIPS) model was set up;3. Senescence-associated ?-galactosidase(SA-?-gal) staining was used to assess the percentage of SA-?-Gal-positive NPCs;4. Cell cycles and cell apoptosis rate were analyzed with flow cytometry, and the growth cures of different passage NPCs were determined by the CCK-8 method.5. The difference expression of growth factors between normal and senescence NPCs was profiled by microarray analysis;6. Confirmation of the selected growth factors was obtained by real-time polymerase chain reaction array analysis.Results:1. Continous subcultured and different concentration of hydrogen peroxide force on normal NPCs could establish RS model and SIPS model, respectively2. In both senescence cell models, the percentages of SA-?-Gal-positive NPCs were more than 70%, the DNA content of G1 were more than 80%, and the cell apoptosis rates were less than 10%;3. Basic fibroblast growth factor(bFGF) and Vascular endothelial growth factor a(VEGF-a) were significant differences between different senescence cells models(p <0.05), moreover, the expression of bFGF in SIPS was signifiant higher than RS(p <0.05). The expression of bFGF in both senescence cells models were signifiant higher than normal group (p<0.05).Conclusion:1. Hydrogen peroxide-induced oxidative stress can promote cells premature senescence;2. Senescence NPCs can upregulate bFGF, and the increase rate is significant higher than RS compared with SIPS. The expression of bFGF may be more regulated than RS compared with SIPS.3. Under different conditions of senescence-induced, the expression of growth factors may differ. bFGF may participate in disc revascularization and repair it.