Ang-2表观遗传学修饰在压力介导的椎间盘退变中的作用机制研究

The mechanism of action of Angiopoietin-2 epigenetic modification in pressure-mediated disc degeneration

目的研究血管生成素-2(Angiopoietin-2,Ang-2)表观遗传学修饰在压力介导的人椎间盘退变中的作用及其作用机制。方法取脊柱侧弯和腰椎间盘突出症患者的椎间盘组织,分离出正常和退变的髓核细胞。将正常及退变髓核细胞放入0 MPa的压力环境下(正常空白组及退变空白组),将正常及退变髓核细胞放入1 MPa的压力环境下(正常压力组及退变压力组),24 h后用逆转录聚合酶链反应(Reverse Transcription-Polymerase Chain Reaction,RT-PCR)、蛋白质印迹法(Western Blot)检测上述4组髓核细胞中靶基因Ang-2、酪氨酸激酶受体-2(Tie-2)、聚集蛋白聚糖(Aggrecan)、Ⅱ型胶原蛋白(CollagenⅡ)和性别决定基因盒转录因子9(sex-determing region of Y chromosome-box transcription factor 9,Sox-9)转录、翻译的表达量。将正常髓核细胞分为3组:空白组、压力组和5-氮杂胞苷组。处理24 h后测量上述指标及MTT法测髓核细胞的活性。结果压力刺激引起髓核细胞中Aggrecan、CollagenⅡ和SOX-9表达水平的减少,但Ang-2和Tie-2表达量增加;退变髓核细胞和正常髓核细胞,以及压力诱导组和一般对照组中,Ang-2的表达都明显增高;DNA甲基化抑制剂可减少压力诱导的髓核细胞中Ang-2、Tie-2的转录和翻译,差异具有统计学意义。MTT检测结果表明,在压力刺激下细胞活性明显下降,DNA甲基化抑制剂延缓压力诱导的髓核细胞活性的下降,差异具有统计学意义。结论以过度压力为特征的髓核细胞外环境发生改变,导致了Ang-2表观遗传修饰状态改变,从而发生过表达,进而诱发了髓核细胞活性的下降以及其细胞外基质的降解,最终加速椎间盘退变。

Objective To study the role and mechanism of Angiopoietin-2(Ang-2) epigenetic modification in pressuremediated human intervertebral disc degeneration. Methods The intervertebral disc tissue of patients with scoliosis and lumbar disc herniation was taken and separated to normal and degenerated nucleus pulposus cells. Put normal and degenerated nucleus pulposus cells under a pressure environment of 0 MPa(normal blank group and degeneration blank group), and put normal and degenerated nucleus pulposus cells under a pressure environment of 1 MPa(normal pressure group and degeneration Pressure group), 24 hours later, RT-PCR and Western Blot were used to detect the transcription and translation expression levels of target genes Ang-2, Tie-2, Aggrecan, collagen Ⅱ and SOX-9 in the 4 groups of nucleus pulposus cells. The normal nucleus pulposus cells were divided into 3 groups: blank group, pressure group,5-azacytidine group. After 24 hours of treatment, the above indicators were measured and the activity of nucleus pulposus cells was measured by MTT method. Results Pressure stimulation caused a decrease in the expression levels of Aggrecan,collagen Ⅱ and SOX-9 in nucleus pulposus cells, but an increase in the expression levels of Ang-2 and Tie-2;degenerative nucleus pulposus cells and normal nucleus pulposus cells, as well as the pressure induction group and the general control group, the Ang-2 The expression is significantly increased;DNA methylation inhibitors can reduce the transcription and translation of Ang-2 and Tie-2 in nucleus pulposus cells induced by stress, and the difference is statistically significant.The MTT test results showed that cell activity decreased significantly under pressure stimulation, and DNA methylation inhibitors delayed the pressure-induced decrease in nucleus pulposus cell activity, and the difference was statistically significant. Conclusion The extracellular environment of the nucleus pulposus characterized by excessive pressure has changed, leading to a change in the epigenetic modification of Ang-2, resulting in overexpression,which in turn induces the decline of nucleus pulposus cell activity and the degradation of its extracellular matrix, and finally accelerates Intervertebral disc degeneration.