Teng J, Fukuda N, Hu W Y, Nakayama M, Kishioka H, Kanmatsuse K
Second Department of Internal Medicine, Nihon University School of Medicine, 173-8610, Tokyo, Japan.
Cardiovasc Res. 2000 Oct;48(1):138-47. doi: 10.1016/s0008-6363(00)00157-7.
The purpose of this study was to develop DNA-RNA chimeric hammerhead ribozyme against transforming growth factor-beta(1) (TGF-beta(1)) mRNA as a gene therapy agent for arterial proliferative diseases.
A 38-base hammerhead ribozyme against rat TGF-beta(1) mRNA, to produce cleavage at the GUC sequence at nucleotide 825 according to the secondary structure of rat TGF-beta(1) mRNA was designed. To enhance its stability, we synthesized a DNA-RNA chimeric ribozyme with two phosphorothioate linkages at the 3'-terminal. We also synthesized a mismatch ribozyme with single base change in the catalytic loop region as a control. These ribozymes were delivered into rat vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats by lipofectin-mediated transfection, and their biological effects were investigated.
According to in vitro cleavage studies, the synthetic ribozyme can cleave the synthetic substrate RNA into two RNA fragments. Chimeric ribozyme significantly inhibited DNA synthesis in VSMC from SHR but not in cells from WKY rats. Mismatch ribozyme showed only a little effect on growth of VSMC from SHR. Chimeric ribozyme significantly inhibited proliferation of VSMC from SHR; in contrast, the proliferation of VSMC from WKY rats was significantly increased by this chimeric ribozyme. Mismatch ribozyme did not affect proliferation of VSMC from either rat strain. Chimeric hammerhead ribozyme to rat TGF-beta(1) dose-dependently inhibited TGF-beta(1) mRNA expression detected by reverse transcription and polymerase chain reaction analysis in VSMC from both rat strains. Chimeric hammerhead ribozyme to rat TGF-beta(1) also dose-dependently inhibited TGF-beta(1) protein production detected by Western blot analysis.
The present results demonstrated that our designed DNA-RNA chimeric hammerhead ribozyme to TGF-beta(1) mRNA might be a useful gene therapy agent for hypertensive vascular diseases.
本研究旨在开发针对转化生长因子-β(1)（TGF-β(1)）mRNA的DNA-RNA嵌合锤头状核酶，作为动脉增殖性疾病的基因治疗药物。
根据大鼠TGF-β(1)mRNA的二级结构，设计了一种针对大鼠TGF-β(1)mRNA的38个碱基的锤头状核酶，可在核苷酸825处的GUC序列产生切割。为提高其稳定性，我们合成了一种在3'末端带有两个硫代磷酸酯键的DNA-RNA嵌合核酶。我们还合成了一种在催化环区域有单个碱基变化的错配核酶作为对照。通过脂质体介导的转染将这些核酶导入自发性高血压大鼠（SHR）和血压正常的Wistar-Kyoto（WKY）大鼠的血管平滑肌细胞（VSMC）中，并研究它们的生物学效应。
根据体外切割研究，合成的核酶可将合成的底物RNA切割成两个RNA片段。嵌合核酶显著抑制SHR来源的VSMC中的DNA合成，但对WKY大鼠来源的细胞无此作用。错配核酶对SHR来源的VSMC生长仅有轻微影响。嵌合核酶显著抑制SHR来源的VSMC增殖；相反，该嵌合核酶使WKY大鼠来源的VSMC增殖显著增加。错配核酶对两种大鼠品系来源的VSMC增殖均无影响。针对大鼠TGF-β(1)的嵌合锤头状核酶在两种大鼠品系来源的VSMC中，通过逆转录和聚合酶链反应分析检测到其剂量依赖性地抑制TGF-β(1)mRNA表达。针对大鼠TGF-β(1)的嵌合锤头状核酶也通过蛋白质印迹分析检测到其剂量依赖性地抑制TGF-β(1)蛋白产生。
目前的结果表明，我们设计的针对TGF-β(1)mRNA的DNA-RNA嵌合锤头状核酶可能是高血压血管疾病的一种有用的基因治疗药物。
