P16INK4a和Fas在椎间盘组织细胞中的表达及意义

目的：分析衰老关键基因p16^INK4a及凋亡相关基因Fas在人类椎间盘退变过程中的表达变化。方法：分别取正常人和腰椎间盘退变患者的髓核及纤维环组织块制作石蜡切片，利用免疫组织化学及免疫荧光法检测p16^INK4a和Fas的表达情况；提取总蛋白及总RNA，利用Western blot及RT-PCR对p16^INK4a和Fas的表达以及视网膜母细胞瘤蛋白（pRb）的磷酸化状态进行分析。结果：p16m在正常人椎间盘髓核及纤维环组织中的表达阳性率分别为6．6％、4．7％，在内破裂椎间盘（IDD）及突出椎间盘（LIDP）组织中的表达分别为44．1％、38．9％和56．1％、46．7％，较正常人椎间盘明显升高（P〈0．05），尤以LIDP中的表达上调显著（P〈0．05）；Fas在正常椎间盘与IDD的髓核及纤维环组织中的表达阳性率均较低。分别为9．9％、8．1％和10．2％、10．9％，在LIDP组织中有相对较高表达阳性率。分别为25．2％和22．0％；Western blot及RT-PCR分析显示，p16^INK4a与Fas在各自蛋白及相应mRNA水平上的表达具有相同的变化趋势：p16^INK4a与Fas极少在同一个椎间盘细胞内表达；随着p16^INK4a表达的升高磷酸化pRb也逐渐减少。结论：p16^INK4a可能参与了椎间盘细胞的衰老过程，是导致椎间盘退变发生和发展的原因之一；Fas表达升高可能是突出椎间盘细胞中的一种继发改变。

Expression and significance of p16INK4a and Fas in lumbar intervertebral discs

Objective:To demonstrate the roles of p16INK4a and Fas in the progression of degeneration of human intervertebral disc(IVD).Method:Sections from normal and symptomatic human discs(IDD and LIDP)were immunostained with an antibody labeling p16INK4a or Fas,and detected by immunohistochemistry and immunofluorescence.In addition,total protein and RNA was isolated from normal and symptomatic human IVD and analysed by Western blot and RT-PCR for p16INK4a and Fas expression on protein and mRNA level.The phosphorization status of pRb was also studied by Western blot.Result:p16INK4a expression in nuclea pulposus and fibrosic annulus of normal IVD was 6.6% and 4.7% respectively,while 44.1%,38.9% and 56.1%,46.7% in IDD and LIPD respectively,which had significant diference compared with those in normal IVD(P<0.05),the upregulation of p16INK4a and Fas was especially higher in LIDP(P<0.05).Fas expression in nuclea pulposus and fibrosic annulus of normal IVD and IDD was 9.9%,8.1% and 10.2%,10.9% respectively.While 25.2% and 22.0% in LIDP.The p16INK4a and Fas expression in the level of both protein and mRNA had the same orientation with both gene rarelly coexpressed in the same cell of IVD.The phaspherized pRb decreased with the rising expression of p16INK4a.Conclusion:p16INK4a may be involved in the senescence of cells in IVD which lead to the progression of degnerative IVD.The rising experssion of Fas may be a reaction secondary to the result of LIPD.