The Effect Of P53and P21Inhibition By Sirna On Senescence And Apoptosis Of NP Cells

The process of intervertebral disc degeneration accompany with the obviouschange of cell biology along with the aging, including the change ofintervertebral disc cell senescence, apoptosis, cell proliferation, cell type, cellnumber, cell density. Degenerative disc cells express senescence associatedβ-galactosidase, especially in the degenerative nucleus pulposus tissue, and theexpression of SA-β-gal was significantly increased along with the grade of discdegeneration. In addition, the senescence of nucleus pulposus cells which wereisolated from the degenerative tissue of intervertebral disc was significantlymore than nucleus pulposus cells that were isolated from the non-degenerativeintervertebral disc. These findings prompted that the senescence of nucleuspulposus cells plays an important role in the process of intervertebral discdegeneration.The intervertebral disc degeneration leads to the rupture of annulus fibrosusand the change of the external environment of the disc. The formation of new blood vessels in the degenerative intervertebral discs improves the hypoxicenvironment of the intervertebral disc, and brings some cytokines. Theproliferative intervertebral disc cells which expose to normoxic condition andcytokines will be more vulnerable to undergoing replicative senescence orstress-induced premature senescence. In short, the senescence of nucleuspulposus cells plays an important role in the process of intervertebral discdegeneration, the cells biological changes of nucleus pulposus cells and theirinteractions in the process of disc degeneration is a much complex. Therefore,the inhibition of the senescence of nucleus pulposus cells may be a potentialbiological treatment for intervertebral disc degeneration.Objective:1. To investigate the feasibility of isolating and culturing nucleus pulposus(NP)cells from rats intervertebral discs by type Ⅱ collagenase and hyaluronidaseand preliminarily identify the phenotype of NP cells by immunocy tochemistry.2. To investigate the feasibility of postponing the senescence of NP cells andalleviating the degeneration of intervertebral disc by inhibiting theexpression of p53, p21through transfection of p53, p21siRNA.Methods:1. The NP cells was isolated and digested by type Ⅱ collagenase andhyaluronidase, and the NP cells were continuous cultured. The expression ofhypoxia inducible factor-1(HIF-1), typeⅡcollagen, matrixmetalloproteinases-2(MMP-2) in NP cells were detected by immunocy tochemistry.2. RT-PCR and Western blot were used to test the silencing rate of NP cellsafter transfection of p53, p21siRNA, SA-β-gal staining is used to test the senescence of NP cells.3. Flow cytometry was used to test the changing of cell cycle of NP cells whichwere transfected with p53, p21siRNA., the growth curve analysis was usedto test the NP cells’ proliferating.Results:1. The NP cells approximately needed12days to ad here, and needed34daysto reach95%confluence in primary culture. After transfer of culture, thecells needed10hours to adhere while the doubling generation time was2.5days. The cultured NP cells express HIF-1α, HIF-1β, MMP2and typeⅡcollagen which are different from annulus fibrosus cells through the stainingof immunocytochemistry.2. The relative expression of mRNA and protein, were assessed by RT-PCRand western blot, of p53and p21were significantly inhibited aftertransfection of p53, p21siRNA. The percentage of SA-β-gal-positive NPcells in the siRNA transfection groups were apparent lower than normalgroup. This result indicates that inhibiting the expression of p53, p21canameliorate the senescence of NP cells.3. Cell cycle analysis showed that G1phase of NP cells in siRNA transfectiongroups were apparent shorter than in normal group, but the S phase of NPcells in siRNA transfection groups were apparent longer than in normalgroup. In addition, the growth curve showed that the growth rate of NP cellsgradually slowed down along with subculture. However, the growth rate ofNP cells accelerated after transfection of p53, p21siRNA. The resultsindicate that inhibiting the expression of p53, p21by siRNA can mediatethe cell cycle of NP cells and promote the proliferation of NP cells.Conclusion: 1. The way of isolating and culturing NP cells by using typeⅡ collagenase andhyaluronidase was successful, and this method improves culture efficiency.HIF-1α, HIF-1β, typeⅡcollagen and MMP2were the characteristicphenotype of NP cells and can be used to identify NP cells.2. The senescence of NP cells can be alleviated or inhibited by silencing of p53and p21.3. Inhibiting the expression of p53, p21can alleviate the senescence of NPcells through mediate the cell cycle, and promote the growth of NP cells, andeven ameliorate the intervertebral disc degeneration. Thus inhibiting theexpression of p53, p21may be a useful and potential therapy for IVDdegeneration.