Gene therapy with antisense MT-MMP oligonucleotide for intimal hyperplasia

反义 MT-MMP 寡核苷酸治疗内膜增生的基因治疗

• 批准号: 09671231
• 负责人: ZEMPO Nobuya
• 金额: $ 1.92万
• 依托单位: Yamaguchi University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671231/
• 关键词: intimal hyperplasia; MT-MMP-1; RT-PCR; antisense oligonucleotide; gene therapy

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.

探讨膜型基质金属蛋白酶-1(MT-MMP1)基因在大鼠颈动脉球囊损伤后的表达，以及反义MT-MMP1寡核苷酸是否抑制培养的SMC的增殖和迁移。逆转录-聚合酶链式反应(RT-PCR)检测MT-MMP1mRNA的表达。MT-MMP1的扩增引物设计如下：正义(5‘-ccc、TAT、GCC、TAC、ATC、CGT、GA-3’)和反义(5‘-TCC、ATC、CAT、CAC、TTG、GTT、AT-3’)。用四甲基偶氮唑盐比色法检测SMC增殖，Boyden小室法检测SMC的迁移(化学侵袭)。MT-MMP1和GAPDH的最适聚合酶链数分别为28和22个。未损伤颈动脉(对照组)MT-MMP1mRNA表达较弱。基因表达在第1天开始升高，第4天达到最大值，之后逐渐下降。用MT-MMP一L聚合酶链式反应产物的放射性除以GAPDH基因产物的相对放射性，对照组第1天为23，第1天80，第4天164，第8天136，第14天54。反义MT-MMIP-L寡核苷酸设计如下：反义1(5‘-AGA CAG GGT CCC CGG CGT CG-3’)和反义2(5‘-AGA 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的增殖和迁移试验。这些结果提示，在球囊损伤后4d，当SMC开始从中膜向内膜迁移时，MT-MMP1mRNA的表达最高。反义MT-MMP1寡核苷酸在体内外均能抑制SMC的增殖和迁移。