Ribozymes that inhibit the production of matrix metalloproteinase 1 reduce the invasiveness of rheumatoid arthritis synovial fibroblasts
Ribozymes that inhibit the production of matrix metalloproteinase 1 reduce the invasiveness of rheumatoid arthritis synovial fibroblasts
复制标题
DOI：
10.1002/art.20186
复制
发表时间：
2004-05-01
影响因子：
--
通讯作者：
Pap, T
中科院分区：
文献类型：
--
作者：
Rutkauskaite, E;Zacharias, W;Pap, T
Objective.研究逆转录病毒介导的靶向基质金属蛋白酶1（MMP-1）的核酶基因转移是否抑制类风湿关节炎滑膜成纤维细胞（RASFs）中MMP-1的产生并降低这些细胞的体内侵袭力。设计MMP-1特异性核酶（RzMMP-1）并将其克隆到pLNSX逆转录病毒载体中。在体外测定MMP-1的切割，并选择最有效的核酶用于进一步研究。用携带RzMMP-1的复制缺陷型病毒或空病毒（模拟）转导RASF。使用具有切割跨位点荧光探针的定量聚合酶链反应来测量MMP-1、MMP-9和MMP-13信使RNA的水平。此外，通过酶联免疫吸附测定法测定细胞培养上清液中MMP-1的蛋白水平。相应地评估用脂多糖（LPS）和肿瘤坏死因子α（TNF α）刺激对MMP-1产生的影响。在RA的SCID小鼠体内模型中分析RzMMP-1转导的、模拟转导的和未转导的RASFs的侵袭性。用RzMMP-1转导RASFs显著降低了RASFs中MMP-1的产生，而不影响其他MMP，如MMP-9和MMP-13。RzMMP-1不仅减少MMP-1的自发产生，而且还阻止LPS和TNF α诱导的MMP-1产生的增加。在RA的SCID小鼠模型中，MMP-1的抑制至少维持2个月，并伴随着RASFs侵袭性的显著降低。核酶的细胞内表达构成了抑制基质降解酶产生的可行工具。单独抑制MMP-1导致RASF对软骨的侵袭显著减少。
Objective. To investigate whether retroviral gene transfer of ribozymes targeting matrix metalloproteinase 1 (MMP-1) inhibits the production of MMP-1 in rheumatoid arthritis synovial fibroblasts (RASFs) and reduces the invasiveness of these cells in vivo.Methods. MMP-1-specific ribozymes (RzMMP-1) were designed and cloned into the pLNSX retroviral vector. Cleavage of MMP-1 was determined in vitro, and the most effective ribozyme was selected for further investigation. RASFs were transduced with replication-deficient viruses carrying RzMMP-1 or with empty viruses (mock). Quantitative polymerase chain reaction with cleavage site-spanning fluorescent probes was used to measure the levels of MMP-1, MMP-9, and MMP-13 messenger RNA. In addition, protein levels of MMP-1 in cell culture supernatants were determined by enzyme linked immunosorbent assay. The effects of stimulation with lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNFalpha) on the production of MMP-1 were assessed accordingly. The invasiveness of RzMMP-1-transduced, mock-transduced, and untransduced RASFs was analyzed in the SCID mouse in vivo model of RA.Results. Transduction of RASFs with RzMMP-1 significantly decreased the production of MMP-1 in RASFs without affecting other MMPs, such as MMP-9 and MMP-13. RzMMP-1 not only reduced the spontaneous production of MMP-1, but also prevented the LPS- and TNFalpha-induced increase in MMP-1 production. Inhibition of MMP-1 was maintained for at least 2 months and was accompanied by a significant reduction of the invasiveness of RASFs in the SCID mouse model of RA.Conclusion. Intracellular expression of ribozymes constitutes a feasible tool for inhibiting the production of matrix-degrading enzymes. Inhibition of MMP-1 alone results in a significant reduction of cartilage invasion by RASFs.