TGF-β1抑制Galectin-3表达调节椎间盘细胞外基质降解及炎性细胞浸润的机制研究

The incidence of low back pain, which is often linked to degenerative changes in the intervertebral disc, is extraordinary high. Low back pain causes an enormous economic burden on individuals, families and society. The commonest strategies are aimed at providing symptomatic relief. Since those treatments can’t completely restore the function of the degenerative intervertebral disc, biological therapy for intervertebral disc degeneration has caused great concern in these years. Intervertebral disc degeneration was characterized by a series of deleterious changes in cellularity that lead to loss of extracellular matrix structure and inflammatory cells infiltration. Our previous studies have demonstrated that inflammatory cytokines play a role in extracellular matrix degradation and inflammatory cells infiltration by inducing the expression of ADAMTS-4、-5 and chemokine CCL3. Recently, studies have shown Galectin-3 participates in the process of intervertebral disc degeneration. The goal of the present study is to investigate the role of Galectin-3 in degenerative disc disease, in particular TGF-β1 regulation of Galectin-3 expression and its role in extracellular matrix degradation and inflammatory cells infiltration. Based on these studies, it is possible to conceive a therapeutic strategy that targets Galectin-3 for the structural preservation of the intervertebral disc.

腰痛在临床上极为常见，给患者家庭及社会造成了沉重负担。导致腰痛的主要病因是椎间盘退变及其继发疾病。目前针对椎间盘退变所引起腰痛的处理以对症治疗为主，疗效欠佳;近年来,生物治疗引起广泛关注。研究证实，椎间盘退变的主要表现为细胞外基质降解及炎性细胞浸润。项目组前期研究发现，细胞因子可通过调节蛋白聚糖酶ADAMTS-4/-5及趋化因子CCL3的表达在细胞外基质降解及炎性细胞浸润中发挥重要作用。近年发现，半乳糖凝集素Galectin-3与椎间盘退变密切相关。预测Galectin-3可通过调节细胞外基质降解及炎性细胞浸润从而在椎间盘退变中发挥作用。为此项目组在前期工作基础上，拟进一步以椎间盘退变为切入点，系统研究和确立转化生长因子β1对Galectin-3的调控机制及其在细胞外基质降解和炎性细胞浸润中的作用及机制，借此为建立通过干预Galectin-3延缓椎间盘退变的治疗策略提供实验依据和理论基础。

本研究旨在对抗炎症因子TGF-β在髓核细胞中对Galectin-3的调节作用进行研究；并对Galectin-3在蛋白聚糖降解及炎性细胞浸润中的作用进行探讨。应用JASPAR等数据库对Galectin-3启动子序列进行分析后发现其启动子内存在9个Smad3的结合位点。接下来我们对TGF-β处理24小时后髓核细胞内Galectin-3启动子活性进行了研究，结果显示，TGF-β能够明显抑制髓核细胞中Galectin-3启动子的活性。DN-ALK5能够阻断TGFβ对Galectin-3启动子活性的抑制作用。功能得到与失去实验进一步证实了Smad3对Galectin-3启动子活性的抑制作用。Smad3与Smad7能够阻断TGF-β对Galectin-3启动子的抑制作用。为进一步研究Smad3在Galectin-3转录调节中的作用。我们对野生型Smad3及Smad3敲除的MEF细胞中Galectin-3的启动子活性进行了进一步的研究。如同我们所预料的一样，野生型Smad3 MEF细胞中，TGF-β可降低Galectin-3启动子的活性，而与之相反，Smad3敲除MEF细胞，TGF-β对Galectin-3启动子活性则没有影响。接下来，我们对人不同等级（Pfirrmann分级）退变髓核组织中提取的髓核细胞中TGF-β对Galectin-3的调节作用进行了研究。结果显示，各等级标本中，TGF-β均可抑制Galectin-3的表达，特别在所有严重退变的标本中，TGF-β均可不同程度地抑制Galectin-3的表达。上述结果提示，TGFβ可通过Smad3通路控制髓核细胞中Galectin-3的表达进而在椎间盘退变中发挥作用。