以MMPs为靶点的SDF-1/CXCR4信号通路在椎间盘软骨终板和髓核退变中的作用和机制研究

The overexpression of matrix metalloproteinases (MMPs), leading to the degradation of extracellular matrix (ECM), is one possible major cause of degenerative cartilage end-plate and nucleus pulposus. However, the molecular mechanism has not yet been studied thoroughly. Cartilage end-plate and articular cartilage are both hyaline cartilage structures. The structural and functional are both similar between chondrocytes in articular cartilage and nucleus pulposus cells. ECM of cartilage end-plate and nucleus pulposus, includes proteoglycan and collagen type II, gradually degrade and the replacement of type I collagen during the process of degeneration. The SDF-1/CXCR4 (stromal cell-derived factor-1/CXC chemokine receptor type 4) pathway targeting MMPs is important in the degeneration of articular cartilage. Blocking this pathway can delay ECM degradation of articular cartilage in vivo, and its role in the endplate cartilage and nucleus pulposus degeneration has not been reported to date. The results of our previous study revealed the existence of SDF-1 and CXCR4 in degenerative discs and their expression levels of degeneration group were significantly higher than those of normal group. We postulated that the SDF-1/CXCR4 pathway also plays a significant role in disc degeneration. For this hypothesis, we plan to investigate the gene and protein expression patterns of SDF-1 and CXCR4 in normal and degenerative discs. Then the expression levels of key MMPs (MMP-1,-3,and-13) as a function of SDF-1/CXCR4 pathway in nucleus pulpsus cells and end plate cartilage cells will be both studied in vitro (cell culture and disc organ culture) to elucidate the intrinsic factor of disc degeneration. The protection or even reverse of degenerative disc will be carried out by inhibition of SDF-1/CXCR4 pathway using RNA interfere technology in vivo. This study will provide profound insights of the mechanism of disc degeneration and thus develop new technology to prevent and treat this disease.

细胞外基质降解是椎间盘软骨终板和髓核退变的主要表现之一，基质金属蛋白酶(MMPs)是其中的关键酶，但是其确切的调控通路尚未完全阐明，阻碍了生物学修复治疗椎间盘退变性疾病的发展。软骨终板与关节软骨一致均为透明软骨，而髓核中的基质成分和细胞生物学特性与关节软骨极为相似。研究表明以MMPs为靶点的SDF-1/CXCR4通路在关节软骨退变中起重要作用，阻断该通路能延缓体内关节软骨ECM的降解，而其在终板软骨和髓核退变中的作用迄今尚未见报道。我们的前期实验发现软骨终板和髓核中均存在SDF-1和CXCR4表达，且退变组的表达水平显著高于正常组。在此基础上，本课题拟采用分子生物学技术、椎间盘器官培养及动物体内实验，研究软骨终板和髓核中以MMPs为靶点的SDF-1/CXCR4通路的调控作用，以及阻断该通路对椎间盘退变的缓解作用。本课题将从新的视角揭示椎间盘退变的发生和发展机制，为其生物学修复提供新的靶点。

椎间盘退变性疾病是临床上最常见的脊柱疾患之一，但是椎间盘退变及自我修复机制仍不甚明了。SDF-1作为一种干细胞迁移因子，其调控MMPs的表达重塑软骨组织以及原位诱导组织再生作用已经得到证实，但是SDF-1在椎间盘组织中的作用及机制仍不得而知。本项目通过采用分子生物学技术、组织培养及动物体内实验，研究椎间盘内SDF-1、CXCR4及MMPs的表达及分布，SDF-1对终板软骨细胞表达MMPs的调控作用，SDF-1对软骨终板组织内细胞外基质的影响，以及动物体内SDF-1/CXCR4信号通路对椎间盘退变所起到的作用。旨在揭示椎间盘中存在以关键MMPs为靶点的SDF-1/CXCR4信号通路，并明确其对椎间盘退变和修复所起到的作用。. 本研究结果证实：1.软骨终板和髓核中均表达SDF-1，终板软骨细胞和髓核细胞表达其受体CXCR4，表达量在退变椎间盘中显著高于正常组；2.SDF-1诱导终板软骨细胞表达CXCR4，MMP-1,-2,-3,-9,-13，并且表达量随着SDF-1浓度而增加，特异性受体阻滞剂AMD 3100可以显著抑制MMPs的表达；3.SDF-1可以重塑终板软骨块中细胞外基质，有助于优化干细胞的迁移通路；4.通过大鼠尾椎纤维环穿刺退变模型，发现尾椎髓核内注射SDF-1可以延缓椎间盘退变，促进椎间盘自我修复。. 综合上述结果，表明椎间盘内存在以关键MMPs为靶点的SDF-1/CXCR4信号通路，该信号通路重塑优化细胞外基质结构，诱导干细胞迁移入椎间盘可能是椎间盘自我修复的重要机制。最近研究报道，椎间盘毗邻的软骨膜和纤维环外层附着部存在干细胞niche，这可能就是椎间盘自我修复中SDF-1/CXCR4信号通路所诱导的干细胞库。我们项目组成功分离了大鼠椎间盘干细胞niche内的干细胞，通过分析表明干细胞具有分化成骨，软骨和脂肪能力。同时，成功构建了SDF-1白蛋白纳米微粒，并通过复合肝素，进一步提高负载带SDF-1的能力和保持生长因子的活性。基于这些研究结果及前期实验，我们项目组获得了国家自然科学基金的连续支持，为进一步揭示椎间盘自我修复机制奠定了坚实的基础，也为将来防治椎间盘退变性疾病提供了新的药物作用靶点。