Zhao Zheng, Li Siyuan, Huang Hui, Fang Jing, Wei Huawei, Xi Yongming
Department of Orthopedics, Affiliated Hospital of Qingdao University, China.
Department of Orthopedics, Shandong Provincial Third Hospital, Jinan, China.
Adv Clin Exp Med. 2020 Jun;29(6):639-647. doi: 10.17219/acem/121509.
Intervertebral disc degeneration (IDD) is characterized by increased proteolytic degradation of the extracellular matrix (ECM), leading to a loss of collagen II and proteoglycan in the nucleus pulposus (NP). Although MMP3 has been reported to play a central role in disc degeneration, it is still unknown whether gene therapy targeting MMP3 can inhibit IDD.
To investigate whether lentivirus-mediated MMP3 knockdown is capable of attenuating IDD. More importantly, we also explored whether combined gene therapy that simultaneously antagonizes MMP3 and overexpresses Sox9 can synergistically inhibit IDD and induce augmented matrix reconstitution in the degenerative NP.
We performed direct injection of lentiviral vectors LV-MMP3-shRNA and/or LV-Sox9 into rabbit lumbar discs. The animals were scanned using magnetic resonance imaging (MRI) at 8, 12 and 24 weeks after the operation. We also evaluated the gene expression and synthesis of NP matrix components, including collagen II, aggrecan and proteoglycan.
The MRI scans showed remarkable needle-puncture-induced progressive IDD in animals injected with PBS or 10^7 viral particles (VP) of the control virus. In contrast, injection of 10^7 VP of LV-MMP3-shRNA or LV-Sox9 substantially inhibited IDD. MMP3 knockdown or Sox9 overexpression stimulated collagen II and aggrecan expression, as well as proteoglycan synthesis. Notably, the injection of a cocktail of LV-MMP3-shRNA and LV-Sox9 (5 × 10^6 VP each) greatly delayed the development of IDD and induced the highest levels of collagen II and proteoglycan production, indicating a synergistic effect in ECM induction.
Our results suggest that gene therapy targeting MMP3 is an efficient way to delay IDD. Combined gene therapy possesses a stronger capacity to induce matrix components in degenerative NP tissue than single-gene delivery.
椎间盘退变（IDD）的特征是细胞外基质（ECM）的蛋白水解降解增加，导致髓核（NP）中Ⅱ型胶原蛋白和蛋白聚糖丢失。尽管已有报道称基质金属蛋白酶3（MMP3）在椎间盘退变中起核心作用，但靶向MMP3的基因治疗是否能抑制IDD仍不清楚。
研究慢病毒介导的MMP3基因敲低是否能够减轻IDD。更重要的是，我们还探讨了同时拮抗MMP3并过表达Sox9的联合基因治疗是否能协同抑制IDD，并在退变的NP中诱导增强的基质重构。
我们将慢病毒载体LV-MMP3-shRNA和/或LV-Sox9直接注射到兔腰椎间盘。术后8、12和24周使用磁共振成像（MRI）对动物进行扫描。我们还评估了NP基质成分的基因表达和合成，包括Ⅱ型胶原蛋白、聚集蛋白聚糖和蛋白聚糖。
MRI扫描显示，注射PBS或10^7病毒颗粒（VP）对照病毒的动物出现明显的针刺诱导的进行性IDD。相比之下，注射10^7 VP的LV-MMP3-shRNA或LV-Sox9可显著抑制IDD。MMP3基因敲低或Sox9过表达刺激了Ⅱ型胶原蛋白和聚集蛋白聚糖的表达以及蛋白聚糖的合成。值得注意的是，注射LV-MMP3-shRNA和LV-Sox9的混合物（各5×10^6 VP）极大地延缓了IDD的发展，并诱导了最高水平的Ⅱ型胶原蛋白和蛋白聚糖产生，表明在ECM诱导中具有协同作用。
我们的结果表明，靶向MMP3的基因治疗是延缓IDD的有效方法。联合基因治疗比单基因递送具有更强的诱导退变NP组织中基质成分的能力。
