高机械张力通过核转录因子κB信号通路促使人髓核细胞退变

High mechanical stretch stress promotes degeneration of the human nucleus pulposus cells through NF-κb signalin_g pathway

目的探讨高机械张力对人髓核细胞的影响及其调控机制。方法收集手术中取出的非退变的髓核组织（Pfirrmann〈Ⅲ级），分离和培养人髓核细胞，在有或无核转录因子κB（NF－κB）的特异性阻断剂（Bay11－7082）预处理的情况下，使用细胞张力加载系统对培养的人髓核细胞加载不同参数的循环机械牵张应力（CMS）；搜集细胞培养液，用酶联免疫方法（Elisa）检测炎症因子分泌情况；搜集加载机械牵张应力后的人髓核细胞，用免疫印迹方法检测人髓核细胞NF－κb/兔P65多克隆抗体信号通路变化，用实时聚合酶链反应方法（rt－PCR）和免疫印迹方法检测椎间盘主要退变指标的mRNA和蛋白质水平表达变化；用白细胞介素（IL）－1β刺激人髓核细胞，用细胞免疫荧光检测P65的变化。结果高振幅（9%、19%）、低频率（0.01 Hz）、长时间（72 h）的机械张力促使人髓核细胞发生退变，而低振幅（3%）、低频率、长时间的机械张力不能促使人髓核细胞发生退变；高振幅、低频率的机械张力加载人髓核细胞24 h后，促进人髓核细胞炎症因子的释放；高振幅、低频率的机械张力可以激活人髓核细胞的NF－κB信号通路，Bay11－7082能够阻断该过程；炎症因子（IL－1β）可以促进人髓核细胞胞质中的P65的磷酸化，促进P65的入核，NF－κB的特异性阻断剂Bay11－7082能够阻断该过程；NF－κB的特异性阻断剂Bay11－7082能明显阻断高机械张力所引起的人髓核细胞的退变趋势，且呈现剂量依赖性。结论高机械张力通过NF－κB信号通路促使人髓核细胞退变。

ObjectiveTo investigate the effect of high mechanical stretch stress（HMS）on human nucleus pulposus cells and its regulatory mechanism.MethodsThe non-degenerated nucleus pulposus tissue （Pfirrmann〈grade Ⅲ） removed from the patient′s surgery was harvested and the human nucleus pulposus cells were isolated and cultured. In the presence or absence of pretreatment with the NF-κB specific blocker Bay11-7082, the cultured human nucleus pulposus cells were loaded cyclic mechanical stretch stress（CMS） with different parameters using the Flexercell system.The cell culture medium was collected and the secretion of inflammatory cytokines was detected by Elisa. The nucleus pulposus cells loaded with cyclic mechanical stretch stress（CMS） was collected, the changes of NF-κB/P65 signal pathway were detected, The mRNA and protein levels′ expression changes were detected by RT-PCR and WB; after human nucleus pulposus cells were exposed to IL-1β, with or without Bay11-7082, the changes of P65 were detected by immunofluorescence.ResultsThose mechanical stretch stress of high amplitude （9%, 19%）, low frequency （0.01 Hz） and long duration （72 h） led to degeneration of human nucleus pulposus cells, while the mechanical stretch stress of low amplitude （3%）, low frequency and long duration could not promote the degeneration process; the mechanical stretch stress of high amplitude（19%）, low-frequency（0.01 Hz） could promote the release of inflammatory cytokines of human nucleus pulposus cells after 24 h duration; high-amplitude, low-frequency mechanical stretch stress could activate the NF-κB signaling pathway in human nucleus pulposus cells, Bay11-7082 could block the process; immunofluorescence showed that IL-1β could promote the phosphorylation of P65 in the cytoplasm of human nucleus pulposus cells and promote the entry of P65 into the cell nucleus process, Bay11-7082 could block those processes; Bay11-7082, the specific blocking agent of NF-κB signaling pathway, could block the degeneration process of human nucleus pulposus cells induced by high cyclic mechanical stretch stress（CMS） in a dose-dependent.ConclusionsHigh cyclic mechanical stretch stress promotes human nucleus pulposus cells degeneration through NF-κB signaling pathway.