基础医学与临床 ›› 2017, Vol. 37 ›› Issue (5): 601-607.

• 研究论文 • 上一篇    下一篇

NF-κB抑制剂减低LPS致兔腰椎间盘髓核细胞锌指蛋白A20表达升高

温亚枫1,易威威2,刘浠2,叶禾2,刘渤2   

  1. 1. 重庆医科大学附属第一医院
    2. 重庆医科大学附属第一医院骨科
  • 收稿日期:2016-10-17 修回日期:2016-11-24 出版日期:2017-05-05 发布日期:2017-04-19
  • 通讯作者: 刘渤 E-mail:boliuuc@163.com
  • 基金资助:
    锌指蛋白A20对髓核细胞炎症反应的调控在椎间盘退变机制中的保护作用研究

NF-κB inhibitor reduces the expression of zinc finger protein A20 in rabbit lumbar intervertebral disc nucleus pulposus cells induced by lipopolysaccharide

  • Received:2016-10-17 Revised:2016-11-24 Online:2017-05-05 Published:2017-04-19

摘要: 目的 观察脂多糖(LPS)刺激退变兔椎间盘髓核细胞前后,锌指蛋白A20(A20)、NF-κB及相关炎性因子的表达及其相互关系。方法 获取正常和退变兔腰椎间盘髓核细胞,分为正常组、退变组、LPS刺激组和NF-κB 抑制剂组,HE染色观察椎间盘髓核及纤维环的形态,免疫组化检测椎间盘A20、NF-κB p65蛋白及Ⅱ型胶原(COL-Ⅱ)的表达。Real-time PCR分别检测各组A20、IL-1β、TNF-α、NF-κB和COL-ⅡmRNA的表达。Western blot观察A20、p65和COL-Ⅱ的表达。ELISA检测细胞培养上清液中IL-1β、TNF-α的表达。结果 退变组髓核细胞数量明显减少,髓核细胞聚集成团,COL-Ⅱ表达下降,A20、p65蛋白表达升高;与正常组相比,退变组A20、TNF-α、IL-1β、p65表达在mRNA及蛋白水平均明显升高,COL-Ⅱ表达降低(p<0.05);LPS刺激组中A20、TNF-α、IL-1β、p65表达较退变组更显著,COL-Ⅱ降低明显(p<0.05),NF-κB 抑制剂组较LPS刺激组A20、TNF-α、IL-1β、p65表达回降,COL-Ⅱ表达回升(p<0.05)。结论 髓核细胞炎性反应与兔椎间盘退变的发生、发展密切相关,其中A20可能发挥了重要作用。

关键词: 关键词 椎间盘退变, 锌指蛋白A20, NF-κB, 炎性反应

Abstract: Objective To observe the expression of zinc finger protein A20(A20), NF-κB and related inflammatory factors before and after lipopolysaccharide (LPS) stimulates degeneration of rabbit intervertebral disc nucleus pulposus cells. Methods The normal and degenerative nucleus pulposus cells were isolated and cultured in vitro,then divided into normal group,degenerative group,LPS stimulation group and NF-κB inhibition group.HE staining observe the morphological changes of nucleus pulposus and annulus fibrosus,immunohistochemistry was used to detect the expression of A20,NF-κB/p65 and COL-Ⅱ.Real-time PCR was employed to analyze the expression of A20,IL-1β,TNF-α,NF-κB and COL-Ⅱ,Western blot was used to observe the A20 protein,p65 and COL-Ⅱexpression in the four groups,and TNF-α,IL-1β in cell supernatant was determined by ELISA. Results The number of nucleus pulposus cells was significantly decreased, aggregation occurs in the degenerative group.COL-Ⅱ was obvious lower and A20, p65 was significantly higher than normal group by immunohistochemical staining. Compared with the normal group,A20,TNF-α,IL-1β,p65 expression was significantly increased and COL-Ⅱ decreased in the mRNA and protein levels in degenerative group.Above indexes change more significant in LPS stimulation group than in degenerative group.The expression of A20, TNF-α, IL-1β, p65 in the NF-κB inhibitor group was lower than that in the LPS group, and the expression of type Ⅱ collagen increased(p<0.05). Conclusions Intervertebral disc inflammatory response is closely related to the development of intervertebral disc degeneration, A20 may play an important role.

Key words: Disc degeneration, Zinc finger protein A20, NF-κB , Inflammatory reaction

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