HtrA1调控髓核细胞ADAMTS-4、5表达机制研究及在椎间盘退变中的意义

Metabolic imbalance of nucleus pulposus cells, which induces acceleration of degradation of functional extracelluar matrix, exists during intervertebral disc degeneration. A disintegrin and metalloproteinase with thrombospondin motifs-4 and 5 (ADAMTS-4, 5) play an important role in this process of degradation of extracellular matrix. Therefore, further research in the regulation of ADAMTS-4 and 5 is particularly important. We previously found that HtrA1 mRNA expression level in nucleus pulposus of patients with disc degeneration was associated with the grade of disc degeneration, and HtrA1 can improve the level of ADAMTS-4 and 5 expression in nucleus pulposus cells. Several other studies have shown that the expression of ADAMTS-4 and 5 may be regulated by NF-κB or MAPK pathway. So we think that the HtrA1 can upregulate the expression of ADAMTS-4 and 5 via NF-κB or MAPK pathway to degrade extracellular matrix and promote disc degeneration. In order to test the hypothesis, we intend to do the following studies: ① investigate the effect of HtrA1 on the expression of ADAMTS-4 and 5 in nucleus pulposus cells using qPCR, Western-Blot, and immunofluorescence methods; ② investigate the mechanism of effect of HtrA1 promoting expression of ADAMTS-4 and 5 through the activation/inhibition of NF-κB or MAPK pathway; ③ verify the effect of blockers of HtrA1-NF-κB/MAPK on the prevention of disc degeneration by in vivo experiments.

椎间盘退变过程中髓核细胞代谢失衡，功能性细胞外基质降解加快，ADAMTS-4、5具有促进胞外基质降解的重要作用。因此，深入研究ADAMTS-4、5的调控机制尤为重要。我们前期发现椎间盘退变患者髓核组织HtrA1的表达与椎间盘退变程度相关，且HtrA1能上调髓核细胞ADAMTS-4、5的表达。文献报道ADAMTS-4、5的表达可受NF-κB及MAPK通路的调控。根据前期研究结合文献报道，我们认为：HtrA1可通过NF-κB或MAPK通路促进ADAMTS-4、5的表达，从而降解细胞外基质，促进椎间盘退变。我们拟：①通过qPCR、Western-blot、免疫荧光等方法证实HtrA1对ADAMTS-4、5表达的影响；②通过NF-κB或MAPK通路的激活/抑制探讨HtrA1促进ADAMTS-4、5表达的信号转导机制；③体内实验验证HtrA1-NF-κB/MAPK信号轴阻断剂对椎间盘退变的防治作用。

本项目的主要研究目的为明确HtrA1在椎间盘退变中的作用，并探索HtrA1介导髓核Aggrecan主要降解酶ADAMTS-4、5表达的潜在机制。项目共分为三个部分：一、不同程度退变的椎间盘髓核组织HtrA1、ADAMTS-4、ADAMTS-5等基因的表达及其相关性。收集临床患者MRI及病理资料，采用RT-PCR及Western-blotting检测髓核组织中HtrA1、ADAMTS-4、5的mRNA及蛋白表达水平，对髓核组织HtrA1、ADAMTS-4、5的表达水平与椎间盘退变程度进行相关性分析。结果显示：椎间盘髓核组织中HtrA1、ADAMTS-4、5的表达水平随退变程度增高而升高，提示HtrA1、ADAMTS-4、5与椎间盘退变程度存在相关性；且髓核组织中HtrA1的表达水平与ADAMTS-4、5的表达水平之间存在线性相关关系。二、HtrA1对髓核细胞ADAMTS-4、5表达的影响。体外试验：分离培养髓核细胞并进行鉴定，采用不同浓度的HtrA1作用于髓核细胞，PCR、Western-blot、ELISA方法检测HtrA1对ADAMTS-4、5表达的影响。体内试验：采用不同浓度的HtrA1注入大鼠椎间盘髓核组织内，提取髓核组织，PCR、Western-blot方法检测HtrA1对ADAMTS-4、5表达的影响。结果显示：HtrA1可促进ADAMTS-5 mRNA及蛋白的表达，以5μg/ml的浓度作用24h效果最为明显，但是对ADAMTS-4的表达并没有明显促进作用。三、机制分析：HtrA1促进髓核细胞ADAMTS-4、5表达的信号转导机制。采用ELISA及Western-blotting 检测相关通路的抑制对HtrA1作用的影响，结果显示：HtrA1能促进ADAMTS-5的表达，NF-κB、ERK、JNK通路分别抑制后，能抑制HtrA1促进ADAMTS-5的表达，说明HtrA1可通过NF-κB、ERK、JNK通路促进ADAMTS-5的表达。本项目的完成明确了HtrA1在椎间盘退变过程中起重要作用，并初步探索了HtrA1介导椎间盘髓核主要基质降解酶ADAMTS-5表达的信号转导机制。