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

我们试图在不降解的情况下延长NFkB寡脱氧核苷酸的释放。首先，我们测试了使用间胶原蛋白将fitc标记的寡核苷酸导入培养细胞的效率。转染HeLa、BHK-21和人舌癌细胞SAS 48h后，未见荧光。在此基础上，构建了日本血凝病毒（HVJ，仙台病毒）包膜载体，该包膜载体可以引入合成的寡核苷酸、蛋白质、多肽和化学药物以及基因。将fitc标记的寡核苷酸通过温和洗涤剂处理和离心将其纳入HVJ包膜载体中。将上述培养细胞与HVJ包膜载体孵育10分钟后，几乎所有细胞核均可见荧光。利用HVJ包膜载体将NFkB诱骗寡核苷酸导入SAS、HeLa等肿瘤细胞，通过抑制NFkB诱导的…More基因的表达，增强了辐照后的细胞凋亡。利用HVJ包膜载体将fitc标记的NFkB诱骗寡核苷酸引入食蟹猴关节间隙，在滑膜和关节软骨处均检测到荧光。然后，在HVJ包络向量上嵌入各种聚合物。首先，不加聚合物，HVJ包膜载体经静脉注射到达脾脏，在小鼠脾脏边缘区检测fitc寡核苷酸。然后，将含有荧光素酶基因的载体用硫酸鱼精蛋白修饰，并将复合物注入小鼠尾静脉。基因表达仅在肺中检测到，在脾脏中未检测到。此外，当混合肝素的HVJ包膜载体直接注射到肌肉或大脑时，基因表达量比不加肝素的增加了约5倍。目前正在研究聚合物对HVJ包膜载体缓释的影响，但我们已经得到了初步的数据表明阳离子聚合物可能会促进HVJ包膜载体的缓释。少

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