Gene therapy with antisense MT-MMP oligonucleotide for intimal hyperplasia

本研究旨在探讨膜型基质金属蛋白酶-1（MT-MMP- 1）基因在球囊损伤大鼠颈动脉中的表达，以及MT-MMP-1反义寡核苷酸是否抑制培养的平滑肌细胞（SMC）的增殖和迁移。逆转录-聚合酶链反应（RT-PCR）检测MT-MMP-1 mRNA的表达。用于扩增MT-MMP-1的引物设计如下：有义引物（5 ′-CCC达特GCC TAC ATC CGT GA-3 ′）和反义引物（5 ′-TCC ATC CAT CAC TTG GTT AT-3 ′）。MTT法检测SMC增殖，Boyden小室法检测SMC迁移（侵袭）能力。MT-MMP-1和GAPDH的最佳PCR循环数分别确定为28和22。正常颈动脉MT-MMP-1 mRNA表达微弱。基因表达在第1天增加，并在第4天达到最大。此后逐渐减少。通过将与MT-MMP-1 PCR产物相关的放射性除以与GAPDH基因产物相关的放射性计算的相对放射性在对照中为23，在第1天为80，在第4天为164，在第8天为136，在第14天为54。反义MT-MMIP-1寡核苷酸被设计用于SMC增殖和迁移测定，如下：反义1（5 '-阿加CAG GGT CCC CGG CGT CG-3'）和反义2（5 '-阿加CAG CAA GGA CCA CTG CC-3'）。通过使用反义寡核苷酸，我们现在正在进行SMC增殖和迁移试验。这些结果表明，MT-MMP- 1 mRNA的表达增加，在最大的球囊损伤后4天，当SMC开始从中膜迁移到内膜。MT-MMP-1反义寡核苷酸的基因治疗在体内外均能抑制SMC的增殖和迁移。

We explored membrane-type matrix metalloproteinase- 1 (MT-MMP- 1) gene expressions in balloon-injured rat carotid artery, and that whether antisense MT-MMP-1 oligonucleotide inhibits cultured smooth muscle cell (SMC) proliferation and migration. The MT-MMP-1 mRNA expressions were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The primers for the amplification of MT-MMP-1 was designed as follows ; sense (5'-CCC TAT GCC TAC ATC CGT GA-3') and antisense (5'-TCC ATC CAT CAC TTG GTT AT-3'). SMC proliferation was measured by MTT assay, and the migration (chemoinvasion ) was assayed by Boyden chamber method. The optimal number of PCR cycles for MT-MMP-1 arid GAPDH was determined as 28 and 22, respectively. The MT-MMP-1 mRNA expression was faint in uninjured carotid arteries (control). The gene expression was increased on day 1 and showed to be maximal on day 4. It was gradually decreased thereafter. The relative radioactivity calculated by dividing the radioactivity associated with MT-MMP-l PCR products by that associated with the GAPDH gene products was 23 in control, 80 on day 1, 164 on day 4, 136 on day 8, and 54 on day 14. The antisense MT-MMIP-l oligonucleotides were designed for the SMC proliferation and migration assays as follows ; antisense 1 (5'-AGA CAG GGT CCC CGG CGT CG-3') and antisense 2 (5'-AGA CAG CAA GGA CCA CTG CC-3'). By using the antisense oligonucleotides, we are now conducting the SMC proliferation and migration assays. These results suggest that MT-MMP- 1 mRNA expression was increased at maximum 4 days after balloon injury when SMCs start to migrate from the media to the intima. Gene therapy with the antisense MT-MMP-1 oligonucleotides might inhibit SMC proliferation and migration in vitro and in vivo.