Development of slow release reagent of NFkB decoy oligodeoxynucleotides for the treatment of rheumatic arthritis

治疗风湿性关节炎的NFkB诱饵寡脱氧核苷酸缓释试剂的研制

基本信息

批准号：
13558109
负责人：
KANEDA Yasufumi
金额：
$ 8.13万
依托单位：
OSAKA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

We attempted to prolong release of NFkB oligodeoxynudeotid.es without degradation. First, we tested the efficiency of introduction of FITC-labeled oligonucleotides into cultured cells using atelocollagen. No fluorescence was detected at 48 hours after the transfer to HeLa, BHK-21 and human tongue cancer cell line SAS. Then, we developed HVJ (hemagglutinating virus of Japan ; Sendai virus) envelope vector which can introduce synthetic oligonucleotides, proteins, peptides and chemical drugs as well as genes. FITC-labeled oligonucleotides were incorporated into HVJ envelope vector by the treatment of mild detergent and centrifugation. Ten minutes after incubation of those cultured cells described above with the HVJ envelope vector, fluorescence was observed in almost all the nuclei of the cells. When NFkB decoy oligonucleotides were introduced into cancer cells such as SAS and HeLa cells using HVJ envelope vector, apoptosis after irradiation was enhanced by the suppression of NFkB-induced … More gene expression. When FITC-labeled NFkB decoy oligonucleotides were introduced into the joint space of Cynomolgus monkeys using HVJ envelope vector, fluorescence was detected at both synovium and articular cartilage. Next, HVJ envelope vector was embedded with various polymers. First, without polymer, HVJ envelope vector reaches spleen by intravenous injection and FITC-oligonucleotides were detected at the marginal zone of mouse spleen. Then, the vector containing luciferase gene was decorated with protamine suifate and the complex was injected into tail vein of mouse. Gene expression was detected exclusively in lung, not in spleen. Furthermore, when HVJ envelope vector mixed with heparin was injected into muscle or brain directly, gene expression was approximately 5 fold increased than that without heparin. The effect of polymers on slow release of the vector is being investigated, but we have got preliminary data suggesting that cationic polymers may enhance slow release of HVJ envelope vector. Less

我们试图延长NFKB寡头氧化物nudeotid.es的释放而不会降解。首先，我们使用atelocollagen测试了将FITC标记的寡核苷酸引入培养细胞中的效率。转移到HELA，BHK-21和人舌癌细胞系SAS后48小时未检测到荧光。然后，我们开发了HVJ（日本的血凝病毒；仙台病毒）包膜载体，该载体可以引入合成寡核苷酸，蛋白质，胡椒粉和化学药物以及基因。通过处理轻度探测器和离心机，将FITC标记的寡核苷酸掺入HVJ包膜载体中。在与HVJ包膜载体描述的那些培养的细胞孵育十分钟后，几乎所有细胞的核都观察到荧光。当NFKB诱饵寡核苷酸使用HVJ包膜载体引入癌细胞，例如SAS和HeLa细胞时，通过抑制NFKB诱导的……更多的基因表达来增强照射后的凋亡。当使用HVJ包膜载体将FITC标记的NFKB诱饵寡核苷酸引入cynomolgus猴子的关节空间时，在滑膜和关节软骨处都检测到荧光。接下来，将HVJ信封矢量嵌入各种聚合物。首先，没有聚合物，HVJ包膜矢量通过静脉注射到达脾脏，并在小鼠臂的边缘区域检测到FITC-寡核苷酸。然后，含有荧光素酶基因的载体装饰有蛋白质，并将复合物注入小鼠的尾静脉中。在肺中仅检测到基因表达，而不是在SLEEN中。此外，当将HVJ包膜矢量与肝素混合直接注入肌肉或大脑时，基因表达大约比没有肝素的hvj载体增加了约5倍。正在研究聚合物对向量缓慢释放的影响，但是我们有初步数据表明阳离子聚合物可能会增强HVJ包膜载体的缓慢释放。较少的