Mechanism On Degeneration Induced By Senescent Nucleus Pulposus Cells

Objective:To set up replicative senescence model and serum-deprived induced senescence model. Observe the senescent NP cells’effect on the normal NP cells and explore its mechanism.Methods:1.Culture normal NP cells with 10%FBS/DMEM-F12 in vitro to set up replicative senescence model.2.Culture the ninth generation of NP cells with DMEM-F12 in vitro to set up serum-deprived induced senescence model.3.Observe the morphology of the NP cells in step 1 and step 2 by microscope. Evaluate NP cells senescence by senescence associated β-galactosidase staining and analysis of cell cycle distribution by flow cytometry. Set up the replicative senescence model and serum-deprived induced senescence model successfully. (In this study proposed the aging rate of 80 percent or higher, G1 phase cells of 80 percent or higher as the standard qualified for NP cells aging model.)4.The expression of TGF-β1 in NP cells was detected by immunofluorescence staining. Detect their expression content of TGF-β1 in the ninth generation of NP cells, replicative senescence model and serum-deprived induced senescence model by QRT-PCR.5.Two different kinds of senescent NP cells were then co-cultured with normal NP cells. Normal NP cells were also cultured alone as controls. At day 7, the morphological characteristics of human NP cells were observed, cellular viability were measured by senescence associated β-galactosidase staining and analysis of cell cycle distribution by flow cytometry. Western-blot method was used to detect their expression content of ERK, Akt, pERK, pAkt of the normal NP cells in the different co-culture systems.Results:1.During the process of setting up inducing replicative senescence model, the morphology of NP cells changed from quasi-circular or star shape to long spindle or irregular shape with the increase of the generations. The size of cells increased and cytoplasm vacuoles became clear gradually. The aging rate by β-galactosidase staining, the percent of G1 phase cells increased gradually. The above two results of the thirteen generation of NP cells were 84.13±4.7% percent,87.59±3.0% percent respectively.2.During the process of setting up serum-deprived induced senescence model at day 7, the morphology changed into long spindle or irregular shape and the size of cells increased compared with the normal NP cells. The aging rate, the percent of G1 phase cells increased significantly. The above two results of the first generation of nucleus pulposus cells by 100umol/L hydrogen peroxide for two hours were 86.70±6.0% percent,88.34±4.4% percent respectively.3.According to the results of QRT-PCR, the expression content of TGF-β1 in two different kinds of senescent NP cells decreased and the difference was statistically significant compared with the normal NP (P<0.05). The expression content between replicative senescence model and serum-deprived induced senescence model had no statistically significant difference(P>0.05).4.In the co-culture system at day 7, we observed an increase in proportions of senescent cells and the percent of G1 phase cells in group A and group B compared with control group(P>0.05). The expression of ERK and Akt is similar in these three groups, while the expression of pERK and pAkt decreased in the normal NP cells of group A and group B compared with control group.Conclusion:1.It was successful to set up replicative senescence model (the thirteen generation of NP cells) by culturing human normal NP cells in vitro, the aging rate and the percent of G1 phase cells increased gradually.2.1t was successful to set up serum-deprived induced senescence model at day 7 with DMEM-F12, the aging rate and the percent of G1 phase cells increased significantly compared with control group which was cultured with 10%FBS/DMEM-F12.3.Human NP cells can express TGF-β1, and the expression content of TGF-β1 in these two senescent models decreased compared with normal NP cells.4.The senescent NP cells could down-regulate the cellular viability of the normal NP cells, the activity of ERK and Akt signal pathway decreased in normal NP cells when there were co-cultured with senescent NP cells.