[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 1alpha(HIF-1alpha, HIF-1beta matrix metalloproteinase 2 (MMP-2), and collagen type II as phenotypic markers were identified 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-beta-galactosidase (SA-beta-gal) staining was used to test the senescence of NP cells, flow 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-1alpha HIF-1beta, MMP-2, and collagen type II. RT-PCR and Western blot showed that the relative expressions of mRNA and protein of p53 and p21 were significantly inhibited in NP cells at passage 35 after transfected with p53 and p21 siRNA. The percentage of SA-Pbetagal-positive NP cells at passage 35 was significantly higher than that at passage 1 (P < 0.001). And the percentage of SA-Pbetagal-positive NP cells in the p53 siRNA transfection group and p21 siRNA transfection group were significantly lower than that in control group (P < 0.001). The flow cytometry showed that the GC1phase of NP cells in p53 siRNA transfection group and p21 siRNA transfection group was significantly 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 significantly 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.


CONCLUSIONS
he 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.
Department of Orthopaedics, Tangdu Hospital, the Fourth Military Medical University, Xi'an Shaanxi, 710038, PR China.
Tao
T
Zhang
Jixian
J
Qian
Zhenwei
Z
Ji
Yunlei
Y
Ma
Zhe
Z
Yun
Chengkui
C
Cai
Xiuchun
X
Qiu
Baoan
B
Ma
D000375:Aging; D000818:Animals; D015153:Blotting, Western; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002478:Cells, Cultured; D020868:Gene Silencing; D016158:Genes, p53; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics* / Q000378:metabolism; D055959:Intervertebral Disc Degeneration / Q000473:pathology; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D054462:p21-Activated Kinases
C497444:Hif1a protein, rat; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C515910:Pak2 protein, rat; D054462:p21-Activated Kinases; D020778:Matrix Metalloproteinase 2