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

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m RNA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m RNA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。

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 S1 PRs was detected by immunohistochemical method. Primary nu-cleus pulposus cells was obtained by collagenas Ⅱ digestion. Real-time PCR and Western-blot were used to assess the S1 PRs in the nucleus pulposus cells. The cytochemistry was used to evaluate the location of S1 PRs 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 pulpo-sus expressed S1PR1/2/3, almost not in the advanced degenerated cells. The real-time PCR showed that the expression of S1 PR decreases in advanced degenerated nucleus pulposus（the normal to the advanced degener-ated 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 S1 PRs 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 S1 P and its receptor may play a role in the degeneration of intervertebral disc.