[沉默p53和p21对延缓髓核细胞衰老的影响]

[Effect of silencing p53 and p21 on delaying senescence of nucleus pulposus cells].

作者信息

Zhang Tao, Qian Jixian, Ji Zhenwei, Ma Yunlei, Yun Zhe, Cai Chengkui, Qiu Xiuchun, Ma Baoan

机构信息

Department of Orthopaedics, Tangdu Hospital, the Fourth Military Medical University, Xi'an Shaanxi, 710038, PR China.

出版信息

Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2012 Jul;26(7):796-802.

PMID:22905613

Abstract

UNLABELLED

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.

CONCLUSION

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.

摘要

未标注

目的髓核（NP）细胞的衰老和死亡是椎间盘退变（IVD）的病理基础。探讨NP细胞的分子表型和衰老机制，确定减轻NP细胞衰老的方法。

方法

从雄性Sprague Dawley大鼠（8 - 10周龄）获取原代NP细胞；通过免疫细胞化学染色鉴定缺氧诱导因子1α（HIF - 1α）、HIF - 1β、基质金属蛋白酶2（MMP - 2）和Ⅱ型胶原作为表型标志物。在NP细胞用p53和p21小干扰RNA（siRNA）转染后，用RT - PCR和蛋白质印迹法检测NP细胞的沉默效果。用衰老相关β - 半乳糖苷酶（SA - β - gal）染色检测NP细胞的衰老情况，用流式细胞术检测细胞周期变化，用生长曲线分析检测NP细胞增殖情况。

结果

免疫细胞化学染色显示NP细胞表达HIF - 1α、HIF - 1β、MMP - 2和Ⅱ型胶原。RT - PCR和蛋白质印迹法显示，用p53和p21 siRNA转染后第35代NP细胞中p53和p21的mRNA和蛋白相对表达量显著受到抑制。第35代SA - β - gal阳性NP细胞的百分比显著高于第1代（P < 0.001）。并且p53 siRNA转染组和p21 siRNA转染组中SA - β - gal阳性NP细胞的百分比显著低于对照组（P < 0.001）。流式细胞术显示，p53 siRNA转染组和p21 siRNA转染组中NP细胞的G1期显著短于对照组（P < 0.05），但p53 siRNA转染组和p21 siRNA转染组中NP细胞的S期显著长于对照组（P < 0.05）。此外，生长曲线显示，转染p53和p21 siRNA后NP细胞生长速率可得到促进。