The Effects Of Oxidative Microenvironmental Stimulus And Cell Consecutive Division In The Senescence Of Human Nucleus Pulposus Cells In Vitro

Objective:To investigate the effects of oxidative microenvironmental stimulus and cell consecutive divison in the senescence of human nucleus pulposus cells in vitro though the changes of expression of matrix metalloproteinase (MMP)-1,-3,-10 and-12. And provide evidence for more effective biological treatment.Methods:Culture human nucleus pulposus cells in monolayer system and establish negative control (NC) group, stress-induced premature senescence (SIPS) group and replicative senescence (RS) group. Use SA-β-Gal staining, PI staining and flow cytometry, Annexin V-FITC/PI and flow cytometry to evaluate the three groups. Using Agilent Whole Human Genome Oligo Microarray (4x44K)to screen for the indexes of diffidence. Measure and compare the expression of MMP-1,-3,-10 and -12, which were chosen in the previous step, by real-time quantitative PCR.Results:1. The senescent cell rates are (4.67±1.83)% in NC Group, (80.92±5.44)% in SIPS Group and (77.08±3.32)% in RS Group. There is no significance between SIPS Group and RS Group (P>0.05).2. The proportions of cells in G1 state are (57.28±2.24)% in NC Group, (84.48±1.08)% in SIPS Group and (88.87±3.15)% in RS Group. There is no significance between SIPS Group and RS Group (P>0.05).3. The cellular apoptosis rates are (2.51±0.45)% in NC Group, (5.40±0.78% in SIPS Group and (8.69±0.92)% in RS Group.4. A lot of indexes of difference in three groups were screened by Agilent Whole Human Genome Oligo Microarray (4×44K). And MMP-1,-3,-10 and -12 were chosen for the following step.5. The result of real-time quantitative PCR shows that the expression of MMPs mentioned above is significantly higher in SIPS Group and RS Group than that in NC Group (P<0.05). The expression of MMPs is also significantly higher in SIPS Group than that of RS Group (P<0.05).Conclusion:1. Both oxidative microenvironmental stimulus and cell consecutive divison can lead to senescence of human NPCs in vitro, resulting in the up-regulation of cell senescence rates, the ratio of cells in G1 phase and the apoptosis rates.2. The biological behavior of human NPCs also changes, with significant differences of many phenotypes expressed.3. Compared with cell consecutive divison, oxidative microenvironmental stimulus has a stronger effect on human NPCs, up-regulating the expression of MMP-1,-3,-10 and -12 more significantly. It indicatesthat oxidative microenvironmental stimulus may play a predominant role in senescence. And biological treatment acting on this point may be more effective.