人退变椎间盘组织中核因子κB信号通路的变化

Expression of NF-κB in a degenerative human intervertebral disc model

目的探讨核因子κB（NF—κB）信号通路在人退变椎间盘中表达的变化及其意义。方法2014年10月至2016年3月收集枣庄矿业集团公司枣庄医院骨科和上海交通大学附属第一人民医院骨科行腰椎手术治疗55例患者的椎间盘组织作为研究对象。将收集的髓核组织分为对照组（未退变椎间盘组织25例）和实验组（退变椎间盘组织30例）。光镜下观察实验组、对照组原代髓核细胞生长的状态；荧光显微镜下观察实验组和对照组NF—κBp65在髓核细胞中的定位情况；采用实时荧光定量聚合酶链反应（RT—PCR）检测目的基因白细胞介素6（IL-6）、基质金属蛋白酶（MMP）、聚集蛋白聚糖酶（ADAMTS）、聚集蛋白多糖（Agg）及Ⅱ型胶原（ColⅡ）的相对定量结果；采用免疫印迹检测核蛋白p65的表达变化；观察抑制NF—κBp65的活性，对ADAMTS-4、MMP-13、Agg及COLⅡ蛋白表达的影响。结果光镜下对照组髓核细胞较实验组髓核细胞细胞体积比较大，胞质丰富，生长速度快。免疫荧光显微镜下实验组髓核细胞p65蛋白主要集中在细胞核中，对照组髓核细胞p65蛋白定位于细胞质。RT—PCR结果显示实验组MMP-1、MMP-3、MMP-13、ADAMTS-4、ADAMTS-5和IL-5的mRNA表达明显高于对照组，而Agg及COLⅡ的mRNA的表达明显低于对照组。免疫印迹检测结果显示核蛋白p65的相对表达量随着髓核组织退变等级的增高逐渐增高；使用BAY11-7082阻断NF—κB信号通路的活性，可以显著的下调ADAMTS-4、MMP-13蛋白的表达，显著上调Agg、COLⅡ蛋白的表达，且随着BAYⅡ-7082浓度的增加，调节作用逐渐加强。结论随着椎间盘退变程度的增加，NF—κB信号通路的活性逐渐升高，可调控基质降解酶过度表达，且在细胞外基质的降解过程起到重要的作用。

Objective To investigate the changes in the expression of NF-κB signaling pathway in human degenerative intervertebral discs. Methods From October 2014 to March 2016,55 nucleus pulposus of surgical patients with degenerative human intervertebral disc were collected for study in Department of Orthopedic Surgey, Hospital of Zaozhuang Mining Corporation, and Department of Orthopedics, Shanghai General Hospital Affiliated Shanghai Jiaotong University, School of Medicine. The collected nucleus pulposus tissues were divided into two groups：experimental group （30） and control group （25）. Cell culture observed normal and degenerative nucleus pulposus cells morphological changes; immunofluoreseence observed NF-κB 1365 changes in the nucleus of nucleus pulposus cells. Real-time PCR was observed changes in aggregated proteoglycans and matrix metalloproteinase gene mRNA. Finally, the use of blockers of nucleus pulposus cells were treated 24 hours, Western blot analysis the changing of p65, ADAMTS-4, MMP-13, aggregate proteoglycans and collagen Ⅱ protein expression. Results Compared with the experimental group, the nucleus pulposus cells in the control group had larger cell volume, abundant cytoplasm and faster growth rate. Cell immunofluorescence show Nondegenerative nucleus pulposus cells p65 protein was mainly localized in the cytoplasm, degeneration of nucleus pulposus ceils p65 protein was mainly concentrated in the nucleus. RT-PCR showed degenerative group of matrix metalloproteinases （ MMP-1, MMP-3, MMP-13 ）, aggrecanase （ADAMTS-4, ADAMTS-5 ） and IL-6 mRNA expression was significantly higher than Nondegenerative group; aggrecan and type Ⅱ collagen expression than those without degeneration group was significantly lower. Expression of nucleus pulposus degeneration in nuclear protein 1365 with the degenerative level increased gradually increased. BAY11-7082 blocked the activity of NF-κB signaling pathway, which could significantly down-regulate the expression of ADAMTS-4 and MMP-13 protein and significantly upregulate the expression of Agg and COL Ⅱ protein. With the increase of BAY11-7082 concentration, gradually strengthened. Conclusion The activation of the NF-KB signaling pathway in a degenerative intervertebral disc is gradually increased, regulating the over-expression of matrix-degrading enzymes. It plays an important role in the degradation of extra-cellular matrix.