| Objective:①Research of annulus fibrosus cell culture conditions and biological characteristics.②Observing the difference of oxidative stress state of the annulus fibrosus cell in different concentrations of H2O2.③Clearing the influence of P38MAPK inhibitor of oxidative stress induced cell biological characteristics of annulus fibrosus cell.④xploring the influence of P38MAPK inhibitor of annulus fibrosus cell gene expression induced by oxidative stress.Methods:①five New Zealand white rabbits (2-3kg, male and female unlimited), separated annulus fibrosus under aseptic conditions, used enzyme digestion method combined tissue block method cultivated with containing15%FBS DMEM/F12(1:1) nutrient solution. When90%cells fused,then using subculture. Used inverted phase contrast microscope to observe cell morphology, trypan blue staining to determine the cell vitality, histopathologic study with toluidine blue and HE staining, MTT method to measure cell proliferation, analyzed the characteristics of the annulus fibrosus cell morphology, vitality, proliferation.②he experiment started when the annulus fibrosus cells attained the second generation. Grouped according to the different concentration of H2O2(0μmol/L、130μmol/L216μmol/L、360μmol/L、600μmol/L、1000μmol/L),0μmol/LH2O2was blank control group, cells in logarithmic phase, different concentrations of H2O2by1h treatment, the original cultures continued to culture48h, by testing, analysed and compared the difference between groups and blank control group on form, energy, value-added, cycle.③The experiment started when the annulus fibrosus cells attained the second generation.Divided into the blank group (make no processing)、the control group (360μmol/L、600μmol/L、1000μmol/L three kinds of concentration of H2O2by1h treatment then changed into the original culture)、the experimental group (With the control group after1h of H2O2treatment,20μmol/L P38MAPK blockers worked30min, then changed into the original culture).After48h,by testing, analysed and compared the difference between groups and blank control group on form, vitality, value-added, age-related-beta-galactose glucoside enzyme activity, cycle.④The experiment started when the annulus fibrosus cells attained the second generation.Divided into the blank group (make no processing)、the control group (600μmol/LH2O2treated1h then changed into the original culture)、the experimental group (With the control group after1h of H2O2treatment,20μmol/L P38MAPK blockers worked30min, then changed into the original culture).After48h, by reverse transcription PCR detected the different expression of P38, P53, and P16, lm-prb mRNA between each group and blank group.Results:①The annulus fibrosus cells began to stick wall after22h, cells present sector, Completely adherent after4d,cells went into exponential phase after4-5d,then appeared subculture after12d. Toluidine blue staining showed nuclei with metachromatic properties. Within three generations, cells could maintain a certain state of cell morphology and growth, over three generations of cells began to change form, appeared vacuoles in cytoplasm and cell proliferation slowed down.②Compared with the blank control group, when the concentration of H2O2were130μmol/L,216μmol/L, annulus fibrosus cell biological characteristics did not have significant change (P>0.05); when the concentration of H2O2were360μmol/L、600μmol/L,1000μmol/L, nucleus pulposus cells appeared aging change:cytoplasm reduced and vacuoles appeared, cell proliferation slowed down, age-related-beta-galactose glucoside enzyme aizen rate increased, cell cycle arrest in G1phase to S phase decreased (P<0.05). With the increasing of H2O2concentration, aging degree increased.③Under the use of P38MAPK,the annulus fibrosus cells with H2O2treatment in concentration of360μmol/L、600μmol/L,the oxidative stress status had improved,but it was invalid under the treatment with1000μmol/L H2O2(P>0.05).④Gene expression was higher in the control group, compared with the blank group and the experimental group data had significant difference (P<0.05). The data of the blank group and experimental group were relatively closed, compared with the data of P38, P16mRNA expression had no significant difference (P>0.05),but the data of expression of P53and lm-prb mRNA were different (P<0.05) Conclusion:①xperiments in intervertebral disc cells, we can choose the second generation of annulus fibrosus cell to experiment,then we can have enough experiments cells at the same time, guarantee the stability of cell activity, and does not affect the result of the experiment.②n the building of a fiber epoxy stress model with the360μmol/L (or more) of the H2O2concentration, so as to lead to annulus fibrosus cell aging ahead of time, cause the change of the annulus fibrosus cell biological characteristics and ensure the oxidative stress model building success.③P38MAPK blockers can intervene in effectively annulus fibrosus cell limited within the scope of the oxidative stress state. When the annulus fibrosus cell oxidative stress level is higher, P38MAPK blockers can’t work, or have little effect.④The experiment can confirm that P38, P16, P53and lm-prb are the important signal of cell aging factor, and exist in the whole P38MAPK signaling pathway, the use of P38MAPK blockers can inhibite the expression of these aging factors on varying degrees, thus delay the process of cell aging, but it can’t restore the annulus fibrosus cells or reversed to oxidative stress state before. |
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