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寡聚脱氧核苷酸的释放。首先，我们用胶原蛋白测试了FITC标记的寡核苷酸导入培养细胞的效率。转移至HeLa、BHK-21和人舌癌细胞系SAS后48h未检测到荧光。在此基础上，我们构建了日本血凝病毒(HJ)包膜载体，该包膜载体可以导入人工合成的寡核苷酸、蛋白质、多肽和化学药物以及基因。通过温和的洗涤剂和离心法将FITC标记的寡核苷酸导入到HVJ包膜载体中。在上述培养细胞与HVJ包膜载体孵育10分钟后，几乎所有的细胞核都观察到了荧光。当NFkB诱骗寡核苷酸通过HvJ包膜载体导入肿瘤细胞如SAS和HeLa时，通过抑制NFkB诱导的…增强了照射后的细胞凋亡更多的基因表达。将FITC标记的NFkB诱骗寡核苷酸通过HVJ包膜载体导入猕猴关节间隙，在滑膜和关节软骨均可检测到荧光。然后，将HVJ包膜载体包埋在各种聚合物中。首先，在没有聚合物的情况下，通过静脉注射将HVJ包膜载体到达脾，并在小鼠脾的边缘区域检测到FITC-寡核苷酸。然后，将含有荧光素酶基因的载体用硫化鱼精蛋白修饰，并将其注射到小鼠尾静脉。基因仅在肺中表达，而在脾中未检测到。此外，将含有肝素的HVJ包膜载体直接注射到肌肉或脑内时，基因的表达比不注射肝素的基因表达增加了近5倍。聚合物对载体缓释的影响正在研究中，但我们已有初步数据表明，阳离子聚合物可能会促进HVJ包膜载体的缓释。较少

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