Analysis Of Inflammation Cytokines Expression Profiling Of Aging Nucleus Pulposus Cells In Humans

Objective:To analyze the inflammation cytokines expression profiling of aging nucleus pulposus cells in humans.Methods:1. Culture human normal nucleus pulposus cells line (Scien Cell4800) in vitro according to the proportion of 1:3 to set up replicative senescence model.2. Culture the eighth generation of nucleus pulposus cells in six holes culture plate by hydrogen peroxide. Treat the nineth generation of nucleus pulposus cells with the action concentration of 100 μmol/L and action time of 2h per day for 4 days to set up stress induced premature senescence model.3. Observe the morphology of every generation of nucleus pulposus cells in step 1 and step 2 by microscope. Evaluate nucleus pulposus cells senescence by senescence associated β-galactosidase staining and PI staining. Set up the replicative senescence model and stress induced premature senescence model successfully. (note:because there was no generally accepted standard of nucleus pulposus cells aging model, the study proposed the aging rate of 80 percent or higher, G1 phase cells of 80 percent or higher as the standard qualified for nucleus pulposus cells aging model.)4. Culture the nineth generation of nucleus pulposus cells, replicative senescence model and stress induced premature senescence model. Detect their expression content of TNF-α,IL-1β,IL-6 and IL-8 by real-time quantification-PCR.Results:1. During the process of inducing replicative senescence model, the morphology of nucleus pulposus 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 (3-galactosidase staining and the percent of G1 phase cells increased gradually. The above two results of the thirteenth generation of nucleus pulposus cells were 81.1 percent,84.5 percent. The difference was statistically significant compared with the normal nineth generation (p<0.05).2. During the process of stress induced premature senescence model, the nineth generation of nucleus pulposus cells was cultured by given concentration of hydrogen peroxide with given action time. The morphology changed from quasi-circular to long spindle and the size of cells increased gradually with more action time and higher action concentration compared with the normal nineth generation. The aging rate, the percent of G1 phase cells increased gradually. The above two results of the nineth generation of nucleus pulposus cells by 100μmol/L hydrogen peroxide for two hours per day with four consecutive days were 80.3 percent,80.6 percent. The difference was statistically significant compared with the normal nineth generation (p<0.05).3. According to the results of RT-PCR, in replicative senescence model, the expression content of TNF-α,IL-1β,IL-6 and IL-8 increased overall and the difference was statistically significant compared with the normal nineth generation (P<0.05). In stress induced premature senescence model, the expression content of TNF-α,IL-1β,IL-6 and IL-8 increased too. The difference was statistically significant compared with the normal nineth generation (P<0.05).Conclusion:1. It was successful to set up replicative senescence model (the thirteenth generation of nucleus pulposus cells) by culturing human normal nucleus pulposus cells line (Scien Cell4800) in vitro.2. It was successful to set up stress induced premature senescence model by acting on the normal nineth generation with 100μmol/L hydrogen peroxide for two hours per day with four consecutive days.3. During the process of both replicative senescence and stress induced premature senescence,the expression content of TNF-α,IL-1β,IL-6 and IL-8 increased overall.4. The expression content of TNF-α,IL-1β,IL-6 and IL-8 in replicative senescence model was similar with that in stress induced premature senescence.