Establishment and evaluation of an in vitro senescence model of mouse nucleus pulposus cells

English Keywords:Nucleus pulposus cells  Tert-butyl hydrogenperoxide  Senescence  Mouse

English Abstract:

  【Abstract】 Objectives: To establish a senescence model of mouse nucleus pulposus cells(NPCs) in vitro. Methods: NPCs were isolated and cultured in vitro from 8-week-old C57BL/6 male mice, and the expression of the NPCs marker protein-aggrecan was detected by cell immunofluorescence(IF) for cell identification. Different concentration gradients of tert-Butyl hydroperoxide(TBHP) of 0μmol/L, 50μmol/L, 100μmol/L, 200μmol/L, 300μmol/L, 400μmol/L, and 500μmol/L were applied to intervene NPCs, and cholecystokinin octapeptide(CCK8) was used to detect the cell activity after 2h and 4h to screen the optimal intervention concentration and time of TBHP. After intervening the P1 generation NPCs with the optimal intervention concentration of TBHP for optimal intervention time, complete medium was applied to culture for further 24h, 48h and 72h, and senescence-associated β-galactosidase staining(SA-β-gal) was used to detect the positive rate of senescent NPCs at each time point and observe the aging changes of NPCs. P2 generation NPCs were divided into control group(complete medium culture) and model group(complete medium culture after intervention with optimal concentration of TBHP for optimal time), real-time quantitative PCR(RT-qPCR) was used to detect the relative cell aging genes of p53, p21 and p16 as well as aggrecan, collagen Ⅱ and matrix metalloproteinase-3(MMP-3), matrix metalloproteinase-13(MMP-13), and a disintegrin and metalloproteinase with thrombospondin motifs-5(ADAMTS-5) mRNA expressions, and western blot(WB) test was applied to detect the protein expressions of aggrecan, collagen Ⅱ, MMP-3, MMP-13, and p53 and p21. Results: Aggrecan protein was highly expressed in the isolated and cultured cells, the NPCs. The results of CCK8 experiment showed that the optimal intervention concentration of TBHP was 100μmol/L and the optimal intervention time was 4h. After 100μmol/L TBHP intervention for 4h and continued culture with complete medium for 24h, 48h and 72h, the positive rate of SA-β-gal of NPCs significantly increased than that in the control group(P<0.05), but there was no significant difference between the three time points of 24h, 48h and 72h(P>0.05). Therefore, complete medium was used to culture for 24h after the optimal intervention concentration of TBHP was added to the model group for optimal intervention time, while the control group was cultured in complete medium for the same time. The results of RT-qPCR showed that compared with the control, the expressions of p53, p21, p16, MMP-3, MMP-13 and ADAMTS-5 mRNA were increased significantly in the model group(P<0.05), while the expressions of aggrecan and collagen Ⅱ mRNA were decreased in the model group(P<0.05). WB detection showed that the protein expressions of MMP-3, MMP-13, p53 and p21 were increased in the model group(P<0.05), but the protein expressions of aggrecan and collagen Ⅱ were decreased(P<0.05). Conclusions: The senescence model of mouse NPCs can be successfully established by TBHP induction, which provides the basis for the study of the pathogenesis of intervertebral disc degeneration.