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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

我们尝试延长 NFkB 寡脱氧核苷酸的释放而不降解。首先，我们测试了使用去端肽胶原将 FITC 标记的寡核苷酸引入培养细胞的效率。转移至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（日本血凝病毒；仙台病毒）包膜载体，可以导入合成的寡核苷酸、蛋白质、肽和化学药物以及基因。通过温和去污剂处理和离心，将 FITC 标记的寡核苷酸整合到 HVJ 包膜载体中。将上述培养的细胞与HVJ包膜载体一起温育10分钟后，几乎在所有细胞的细胞核中都观察到荧光。当使用 HVJ 包膜载体将 NFkB 诱饵寡核苷酸引入 SAS 和 HeLa 细胞等癌细胞时，通过抑制 NFkB 诱导的基因表达，增强了辐射后的细胞凋亡。当使用 HVJ 包膜载体将 FITC 标记的 NFkB 诱饵寡核苷酸引入食蟹猴的关节间隙时，滑膜和关节软骨均检测到荧光。接下来，HVJ 包膜载体嵌入各种聚合物。首先，在没有聚合物的情况下，HVJ包膜载体通过静脉注射到达脾脏，并在小鼠脾脏的边缘区检测到FITC-寡核苷酸。然后，用硫酸鱼精蛋白修饰含有荧光素酶基因的载体，并将复合物注射到小鼠尾静脉中。基因表达仅在肺中检测到，而不是在脾脏中检测到。此外，当与肝素混合的HVJ包膜载体直接注射到肌肉或大脑中时，基因表达比没有肝素的情况增加约5倍。聚合物对载体缓慢释放的影响正在研究中，但我们已经得到初步数据表明阳离子聚合物可以增强HVJ包膜载体的缓慢释放。较少的