NF-κB信号通路调控Asporin在椎间盘退变中的作用及分子机制研究

The intervertebral disc degeneration (IVDD) is a common disorder with high morbidity, but its molecular pathogenesis and underlying mechanisms have not been fully elucidated. Our previous studies have found that the activation and abnormal expression of NF-κB signaling pathway were associated with IVDD. In our preliminary experiments, the mechanisms of IVDD were assessed by gene microarray, gene ontology, and pathway analysis; we have found that the NF-κB signaling pathway and Asporin play an important role in IVDD. Moreover, we validated these results in clinical specimens using qRT-PCR. Further analysis has revealed that two NF-κB binding sites exist in the promoter sequence of Asporin gene. Based on previously reports and our preliminary data, we hypothesized that NF-κB regulates Asporin, which is involved in IVDD development and progression. The present project is designed to investigate the role of NF-κB and Asporin in IVDD both in vitro and in vivo, using state-of-the-art cell and molecular biology techniques and cutting-edge IVDD models. Upon completion of the proposed studies, we will provide a basis for future research to develop effective and safe therapy for IVDD.

椎间盘退变是临床常见病，其机制仍未完全阐明，如何延缓椎间盘退变是国内外基础和临床研究的热点。本课题组前期发现NF-κB信号活化和异常表达与椎间盘退变相关。前期实验采用基因芯片分析了正常和退变的椎间盘表达谱，通过信号通路分析，捕获了NF-κB信号通路和Asporin作为分子靶点。进一步分析发现Asporin启动子序列中存在2个NF-κB的结合位点。本研究的中心假说是NF-κB信号通路在转录水平调控Asporin的表达，参与椎间盘退变。因此，本研究拟采用基因转染、Westernblot、荧光定量PCR等方法在细胞和临床标本中探讨NF-κB信号通路调控Asporin启动子的表达，进而控制Asporin蛋白及mRNA合成，参与椎间盘退变的作用及分子机制，并用动物模型加以验证，评估它们是否可以成为有效延缓椎间盘退变的有效靶标。我们相信本课题的研究成果将为研发有效及安全的治疗手段提供理论与实验基础。

椎间盘退变是临床常见病，其机制仍未完全阐明，如何延缓椎间盘退变是国内外基础和临床研究的热点。本课题组前期发现NF-κB信号活化和异常表达与椎间盘退变相关。前期实验采用基因芯片分析了正常和退变的椎间盘表达谱，通过信号通路分析，捕获了NF-κB信号通路和Asporin作为分子靶点。进一步分析发现Asporin启动子序列中存在2个NF-κB的结合位点。.本课题主要研究包括三方面。一：Asporin基因在椎间盘髓核细胞中的表达情况。1.在退变的人椎间盘髓核组织中Asporin表达增加，且Asporin表达量与退变程度正相关； 2.针刺法能成功制作兔椎间盘退变模型；3.针刺法制作的兔椎间盘退变模型中，随着退变加重，Asporin表达逐渐增加。二、Asporin基因在人髓核细胞中的调控机制。1.炎症因子（IL-1β）能够显著促进人髓核细胞Asporin表达增加；2.炎症因子（IL-1β）通过NF-κB/P65信号通路促进Asporin启动子活性的增加；3.Asporin启动子序列中存在1个NF-κB的结合位点，相对于转录起点的位置及相对评分为：TGTGTTTTCCA,-31/-41,8.019；三Asporin基因在椎间盘髓核细胞中功能。1. Asporin蛋白能降低TGF-β1对人髓核细胞aggrecan、collagenⅡ表达、分泌的促进作用；2. Asporin蛋白能增强IL-1β对人髓核细胞aggrecan、collagenⅡ表达、分泌的抑制作用。.本课题，围绕着Asporin基因在人髓核细胞中的调控机制和在椎间盘退变中的作用，采用Western Blot、免疫组化、病毒沉默、质粒高表达、荧光素酶双报告基因等分子生物学技术，得到如下结论：1. Asporin基因在退变的髓核组织中高表达，且表达量与退变程度正相关；2. IL-1β通过NF-κB信号通路调节Asporin基因的表达；3. Asporin蛋白通过拮抗TGF-β1的作用，促进人髓核细胞退变；4. Asporin蛋白通过增强IL-1β的作用，促进人髓核细胞退变。