Joint Study on the Therapy of Diseases by Gene Transfer

我们研制了一种高效的基因载体HVJ-脂质体，试图利用这种载体来预防心肌梗死血管成形术后的再狭窄，这是心肌梗死治疗的一个障碍。我们研究了AS-ODNs对再狭窄的治疗作用。我们首先通过HVJ脂质体将AS-PCNA和AS-cdc 2激酶导入球囊损伤血管。两种AS-ODNs共转染后，PCNA和cdc 2激酶mRNA的表达均被抑制至检测不到的水平。然后将含有15 μ M AS-PCNA和AS-cdc 2激酶的HVJ-脂质体注射到球囊损伤的大鼠颈动脉中。这种处理完全抑制了新生内膜的形成，而正义寡核苷酸则没有效果。结果表明，AS对新生内膜形成的抑制作用可持续2周，转染后可持续8周。 关于我们 AS-PCNA与cdc 2激酶或AS-cdc 2激酶与cdk 2激酶联合应用对新生内膜形成的抑制作用最强。然后，我们开发了通过引入单个分子来抑制细胞增殖的新策略。PCNA、cdc 2激酶和一些原癌基因的转录都被一个共同的转录因子E2 F激活，E2 F识别的共有DNA序列是TTTCGCGC。我们研究了E2 F诱饵对预防再狭窄的作用。采用HVJ-脂质体将E2 F诱饵导入球囊损伤的大鼠颈动脉。我们的研究结果表明，在血管成形术后2周，针对E2 F的诱骗物显著抑制新生内膜形成。在3 μ M的剂量下，诱骗物ODN与单独用HVJ-脂质体处理的血管或错配诱骗物ODN处理的血管相比，抑制新生内膜形成约80%。然后，我们用一氧化氮合酶cDNA抑制血管新生内膜的形成，用HVJ脂质体将一氧化氮合酶导入血管壁抑制血管新生内膜的形成至少一个月。这些策略将能够应用于治疗人类再狭窄。少

We developed efficient gene delivery system called HVJ-liposomes, Using this delivery system, we attempted to prevent restsnosis after angioplasty which is a barrier to the therapy of myocardial infarction. We examined the effect of AS-ODNs for the therapy of restenosis. First we introduced AS-PCNA and AS-cdc2 kinase into balloon injured vessels by HVJ-liposome. Cotransfer of both AS-ODNs resulted in the simultanuous inhibition of PCNA and cdc2 kinase mRNA expression to undetectable levels. Then HVJ-liposome containing 15 uM AS-PCNA and AS-cdc2 kinase was injected into balloon injured rat carotid artery. Neointima formation was completely inhibited by this treatment, whereas the sense-ODNs had no effect. The blockade of neointima formation by these AS continiued for 2 weeks by a single intraluminal administration and the inhibitory effect lasted for a period of 8 weeks after transfection.We further examined effect of AS-ODN against various cell-cycle regulators on neointima formation. … More The combination of AS-PCNA and cdc2 kinase or AS-cdc2 kinase and cdk2 kinase were most effective for the prevention of neointima formation. Then, we developed new strategy to inhibit cell proliferation by the introduction of a single molecule. The transcription of PCNA,cdc2 kinase and some protooncogene were activated by a common transcription factor, E2F,and the consensus DNA sequence recognized by E2F is known to be TTTCGCGC.We investigated the effect of double-stranded ODN designated as competitor of E2F on the prevention of neointima formation. We examined the effect of E2F decoy on the prevention of restenosis. E2F decoy was introduced into balloon injured rat carotid artery by HVJ-liposome. Our results demonstrated a marked suppression of neointima formation at 2 weeks sfter angioplasty by the decoy against E2F.At a dosage of 3 uM,decoy ODN inhibited neointima formation by approximately 80 % compared to vessels treated with HVJ-liposome alone or mismatched decoy ODN-treated vessels. Then, we employed NO synthase cDNA to inhibit neointima formation.NOS introduced into blood walls by HVJ-liposomes inhibited neointima formation for at least one month. These strategies will be able to be applied for the treatment of restenosis in humans. Less