1-磷酸鞘氨醇受体在不同退变程度髓核组织中的表达变化

Expression of sphingosine 1-phosphate receptors in degenerative nucleus pulposus with different degrees

中文关键词:  椎间盘退变  1-磷酸鞘氨醇受体  髓核细胞

英文关键词:Intervertebal disk degeneration  Sphingosine 1-phosphate receptors  Nucleus pulposus cells

中文摘要:

  【摘要】 目的：比较不同退变程度人椎间盘髓核组织中3种1-磷酸鞘氨醇受体（S1PR1/2/3）表达水平的差异，探讨椎间盘中S1PR表达水平与椎间盘退变的关系。方法：收集腰椎间盘退行性病变患者手术切除的椎间盘组织，其中轻度退变（Pfirrmann分级Ⅳ级）22例，严重退变（Pfirrmann分级Ⅴ级）14例；同时取6例无椎间盘退变患者（单纯腰椎椎体骨折，Pfirrmann分级Ⅱ级）手术切除的椎间盘组织作为对照组；通过HE染色以及Saf-O染色观察不同退变程度椎间盘的组织学变化，免疫组化检测不同退变程度组织中的S1PR表达水平；Ⅱ型胶原酶消化分离提取原代髓核细胞，通过Real-time PCR、Western-bolt检测不同退变程度椎间盘髓核细胞中S1PR的表达水平，并通过细胞免疫化学方法对S1PR进行定位。结果：HE染色及Saf-O染色结果显示退变椎间盘的纤维环出现破损，髓核细胞形成明显的集落，细胞外基质减少。免疫组化结果显示正常和轻度退变的髓核组织中3种受体（S1PR1/2/3）都有表达，严重退变的组织中表达极弱；Real-time PCR结果显示对照组髓核细胞中 S1PR1/2/3 的mRNA表达水平分别是严重退变组的5.34±0.52倍、7.25±0.04倍、1.92±0.06倍，轻度退变组S1PR1/2/3 的mRNA表达水平分别是严重退变组的4.35±2.45倍、4.96±3.44倍、2.19±0.82倍；Western-blot发现对照组和轻度退变组髓核细胞中S1PR1/2/3均有表达，严重退变组表达水平较低；免疫细胞化学显示S1PR主要集中在髓核细胞的细胞质和细胞膜上。结论：髓核组织中主要表达S1PR1/2/3，在严重退变的髓核组织和细胞中其表达水平明显下降，S1P及其受体可能参与椎间盘髓核组织的退变过程。

英文摘要:

  【Abstract】 Objectives: To compare the expression of sphingosine 1-phosphate receptors(S1PRs) in degenerative nucleus pulposus with different degrees, and to analyze the relationship between the expression level and the degeneration. Methods: 22 mild degenerative intervertebral discs(Pfirrmann grade Ⅳ) and 14 advanced degenerative(Pfirrmann grade V) discs were collected as samples, 6 normal samples(Pfirrmann grade Ⅱ, simple vertebral fracture) were used as controls. HE staining and Saf-O staining was used to show the histological changes of degeneration. The expression of S1PRs was detected by immunohistochemical method. Primary nucleus pulposus cells was obtained by collagenas Ⅱ digestion. Real-time PCR and Western-blot were used to assess the S1PRs in the nucleus pulposus cells. The cytochemistry was used to evaluate the location of S1PRs in nucleus pulposus cells. Results: HE and Saf-O staining showed intervertebral disk with advanced degeneration: fracture appeared in annulus fibrosus, cells bind together into clones in nucleus pulposus and extracellular matrix. The immunohistochemical result showed that normal and mild degenerated nucleus pulposus expressed S1PR1/2/3, almost not in the advanced degenerated cells. The real-time PCR showed that the expression of S1PR decreases in advanced degenerated nucleus pulposus(the normal to the advanced degenerated was 5.34±0.52-flod， 7.25±0.04-flod， 1.92±0.06-flod, the mild degenerated to the advanced was 4.35±2.45-flod, 4.96±3.44-flod, 2.19±0.82-flod). Western-blot result showed that NP cells form control and mild degeneration expressed S1PR1/2/3, while much weak in the advanced degenerated NP cells. The cytochemistry showed that S1PRs focused in the membranes and cytoplasm of cells. Conclusions: The intervertebral disc mainly expresses S1PR1/2/3, and the expression decreases in the advanced degenerative nucleus pulposus. The S1P and its receptor may play a role in the degeneration of intervertebral disc.