| 摘要: |
| 目的 探讨野生型p53诱导的蛋白磷酸酶1(Wip1)在人椎间盘髓核细胞放射性损伤DNA修复中的可能作用,为椎间盘退行性变的临床诊治提供参考。方法 取人椎间盘髓核细胞体外培养,小干扰(siRNA)技术干扰细胞Wip1表达,放射性照射(4、10、15、25 Gy)髓核细胞,采用彗尾实验观察DNA损伤及损伤修复反应(DNA damage repair, DDR)情况,并与未干扰Wip1髓核细胞作对照；采用免疫共沉淀(Co-IP)技术检测Wip1潜在结合蛋白；根据筛选结果采用实时定量RT-PCR技术检测病变椎间盘组织Wip1及潜在结合蛋白的表达情况。结果 彗尾实验表明：干扰Wip1表达后,25 Gy剂量射线导致髓核细胞DNA损伤的修复并不受影响,但DDR持续激活,对照组在照射后24 h已经检测不到DNA损伤修复的标识分子,但siRNA干扰Wip1表达组细胞照射后48 h仍可检测到标识分子。Co-IP实验表明：放射性照射正常对照细胞后,Wip1与Bmi1在细胞核内分布重合,而且聚集在DNA损伤位点,而siRNA抑制Wip1表达后,这种结合随即消失。实时定量RT-PCR结果表明：与正常椎间盘组织相比,椎间盘退变组织Wip1、Bmi1基因表达下降,且两者呈正相关(P<0.05)。结论 Wip1通过调控DDR时限参与了人椎间盘髓核细胞放射性损伤DNA修复,Bmi1可能参与此过程。 |
| 关键词: 椎间盘退行性疾病 辐射损伤 DNA修复 Wip1 Bmi1 |
| DOI： |
| 投稿时间：2013-01-05修订日期：2013-06-04 |
| 基金项目:国家自然科学基金(81171778,31171086). |
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| Wild-type p53-induced phosphatase 1 combined with Bmi1 in repairing ion radiation-induced DNA damage in nucleus pulposus cells |
| YU Lei1,HE Jiang-tu2,WANG Qin-wan2,YU Yong-chun2,PAN Qiu-hui2,LI Jing-hua2* |
(1. Department of Clinical Laboratory, The Tenth Peoples’ Hospital of Shanghai, Tongji University, Shanghai 200072, China
2. Central Laboratory, The Tenth Peoples’ Hospital of Shanghai, Tongji University, Shanghai 200072, China
*Corresponding author.) |
| Abstract: |
| Objective To investigate the possible role of wild-type p53-induced phosphatase 1 (Wip1) in repairing radiation-induced DNA damage in nucleus pulposus (NP) cells, so as to provide reference for treatment of intervertebral disc degeneration. Methods Human NP cells were cultured in vitro and the expression of Wip1 was knocked down by small interfering (siRNA) technology; the NP cells were exposed to radiation (4,10,15 and 25 Gy). DNA damage and repair were observed by comet assay, and the results were compared with NP cells not treated by siRNA. Co-immunoprecipitation (Co-IP) was used to detect the potential binding protein of Wip1, and based on the screening results, qRT-PCR was used to examine Wip1 expression and expression of potential binding protein. Results Comet assay showed that the repair of DNA damage in Wip1 knockdown NP cells was not affected when exposed to 25 Gy radiation, but DNA damage repair was persistently reactivated. The marker molecules of DNA damage repair were not detectable 24 h after radiation in the control group, but they could be detected 48 h after radiation in Wip1 knockdown group. Co-IP results showed that the distribution of Wip1 and Bmi1 coincided in the nuclei of normal cells exposed to radiation, aggregating at the DNA damage site, and the coincidence disappeared after knockdown of Wip1. qRT-PCR results showed that, compared with the normal intervertebral tissues, degenerated intervertebral tissues had significantly decreased Wip1 and Bmi1 expression, and the expression of Wip1 was positively correlated with Bmi1 expression (P<0.05). Conclusion Wip1 plays an important role in the DNA damage repair of NP cells by regulating DNA damage repair phase, and Bmi1 may also participate in this process. |
| Key words: intervertebral disc degeneration radiation injuries DNA repair Wip1 Bmi1 |
