EFFECT OF SILENCING p53 AND p21 ON DELAYING SENESCENCE OF NUCLEUS PULPOSUS CELLS

Objective The senescence and death of nucleus pulposus(NP) cells are the pathologic basis of intervertebral disc degeneration(IVD).To investigate the molecular phenotypes and senescent mechanism of NP cells,and to identify the method of alleviating senescence of NP cells.Methods The primary NP cells were harvested from male Sprague Dawley rats(8-10 weeks old);the hypoxia inducible factor 1α(HIF-1α),HIF-1β,matrix metalloproteinase 2(MMP-2),and collagen type II as phenotypic markers were identi ed through immunocytochemical staining.RT-PCR and Western blot were used to test the silencing effect of NP cells after the NP cells were transfected with p53 and p21 small interference RNA(siRNA).Senescence associated-β-galactosidase(SA-β-gal) staining was used to test the senescence of NP cells,ow cytometry to test the change of cell cycle,the growth curve analysis to test the NP cells proliferation.Results Immunocytochemical staining showed that NP cells expressed HIF-1α,HIF-1β,MMP-2,and collagen type II.RT-PCR and Western blot showed that the relativeffexpressions of mRNA and protein of p53 and p21 were signi cantly inhibited in NP cells at passage 35 after transfected with p53 and p21 siRNA.The percentage of SA-β-gal-positive NP cells at passage 35 was signi cantly higher than that at passage 1(P 0.001).And the percentage of SA-β-gal-positive NP cells in the p53 siRNA transfection group and p21 siRNA transfection group were signi cantly lower than that in control group(P 0.001).The ow cytometry showed that the G1phase of NP cells in p53 siRNA transfection group and p21 siRNA transfection group was signi cantly shorter than that in control group(P 0.05),but the S phase of NP cells in p53 siRNA transfection group and p21 siRNA transfection group were signi cantly longer than that in control group(P 0.05).In addition,the growth curve showed that the growth rate of NP cells could be promoted after transfection of p53 and p21 siRNA.Conclusion The senescence of NP cells can be alleviated by silencing of p53 and p21.The effect of alleviating senescence can even ameliorate the progress of IVD and may be a useful and potential therapy for IVD.